Devices for Detecting Renal Disorders

ABSTRACT

Devices for diagnosing, monitoring, or determining a renal disorder in a mammal are described. In particular, devices for diagnosing, monitoring, or determining a renal disorder using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application Ser. No. 61/327,389, filed Apr. 23, 2010, and U.S. provisional application Ser. No. 61/232,091, filed Aug. 7, 2009, each of which is hereby incorporated by reference in its entirety, and is related to U.S. patent application Ser. Nos. [Not Yet Assigned], entitled Methods and Devices for Detecting Obstructive Uropathy and Associated Disorders, Computer Methods and Devices for Detecting Kidney Damage, Methods and Devices for Detecting Kidney Damage, Methods and Devices for Detecting Kidney Transplant Rejection, Methods and Devices for Detecting Diabetic Nephropathy and Associated Disorders, and Methods and Devices for Detecting Glomerulonephritis and Associated Disorders, Attorney Docket Nos. 060075-, filed on the same date as this application, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention encompasses devices for diagnosing, monitoring, or determining a renal disorder in a mammal. In particular, the present invention provides methods and devices for diagnosing, monitoring, or determining renal disorders in a mammal using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.

BACKGROUND OF THE INVENTION

The urinary system, in particular the kidneys, perform several critical functions such as maintaining electrolyte balance and eliminating toxins from the bloodstream. In the human body, the pair of kidneys together process roughly 20% of the total cardiac output, amounting to about 1 L/min in a 70-kg adult male. Because compounds in circulation are concentrated in the kidney up to 1000-fold relative to the plasma concentration, the kidney is especially vulnerable to injury due to exposure to toxic compounds.

Renal disorders and disease are serious conditions that generally affect the function of the kidney. The disorders discussed herein may arise from a variety of causes, including intrinsic disease processes, such as inflammation and necrosis of the kidney. In addition, renal disorders may also arise from secondary sources including drugs that are toxic to the kidneys and alternative disease states that cause secondary adverse effects on the kidney, such as diabetes and hypertension. Prevention of renal disorders is largely dependent on early diagnosis of the condition. Existing diagnostic tests such as BUN and serum creatine tests typically detect only advanced stages of kidney damage. Other diagnostic tests such as kidney tissue biopsies or CAT scans have the advantage of enhanced sensitivity to earlier stages of kidney damage, but these tests are also generally costly, slow, and/or invasive.

A need exists in the art for a fast, simple, reliable, and sensitive method of detecting obstructive uropathy or an associated disorder. In a clinical setting, the early detection of kidney damage would help medical practitioners to diagnose and treat kidney damage more quickly and effectively.

SUMMARY OF THE INVENTION

The present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder in a mammal. In particular, the present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.

In one aspect, the present invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising six antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.

In another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol. It is also recognized that the assay device may include combinations of 6, 10, or 16 antibodies with antigenic determinants corresponding to the analytes disclosed herein.

In another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising: (a) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol; (b) three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; (c) three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and (d) three or more indicators, wherein each of the indicators is attached to one of the detection antibodies.

In a further aspect, the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal, where the kit includes: (a) an assay device having a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; and (b) a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.

In yet another aspect, the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal, where the kit includes: (a) an assay device having (i) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; (ii) three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; (iii) three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and (iv) three or more indicators, wherein each of the indicators is attached to one of the detection antibodies; and (b) a collection apparatus suitable for collecting a sample bodily fluid from the mammal.

In still another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers having sixteen antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.

In a further aspect, the invention encompasses a platform for diagnosing, monitoring, or determining a renal disorder in a mammal, the platform comprising at least 6 antibodies selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.

Other aspects and iterations of the invention are described in more detail below.

DESCRIPTION OF FIGURES

FIG. 1 depicts four graphs comparing (A) the concentrations of alpha-1 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (B) the concentrations of beta-2 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (C) the concentrations of NGAL in the urine of normal controls, kidney cancer patients, and patients with other cancer types; and (D) the concentrations of THP in the urine of normal controls, kidney cancer patients, and patients with other cancer types.

FIG. 2 shows the four different disease groups from which samples were analyzed, and a plot of two different estimations on eGFR outlining the distribution within each group.

FIG. 3 is a number of scatter plots of results on selected proteins in urine and plasma. The various groups are indicated as follows—control: blue, AA: red, DN: green, GN: yellow, OU: orange. (A) A1M in plasma, (B) cystatin C in plasma, (C) B2M in urine, (D) cystatin C in urine.

FIG. 4 depicts the multivariate analysis of the disease groups and their respective matched controls using plasma results. Relative importance shown using the random forest model.

FIG. 5 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish disease samples vs. normal samples. Disease encompasses analgesic abuse (AA), glomerulonephritis (GN), obstructive uropathy (OU), and diabetic nephropathy (DN). Normal=NL.

FIG. 6 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish disease (AA+GN+ON+DN) samples vs. normal samples from plasma (A) and urine (B and C).

FIG. 7 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. normal samples. Abbreviations as in FIG. 4.

FIG. 8 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. normal samples from plasma (A) and urine (B and C).

FIG. 9 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. diabetic nephropathy samples. Abbreviations as in FIG. 4.

FIG. 10 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. diabetic nephropathy samples from plasma (A) and urine (B and C).

FIG. 11 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish glomerulonephritis samples vs. analgesic abuse samples. Abbreviations as in FIG. 4.

FIG. 12 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish glomerulonephritis samples vs. analgesic abuse samples from plasma (A) and urine (B and C).

FIG. 13 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish obstructive uropathy samples vs. analgesic abuse samples. Abbreviations as in FIG. 4.

FIG. 14 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish obstructive uropathy samples vs. analgesic abuse samples from plasma (A) and urine (B and C).

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that a multiplexed panel of three or more, six or more, and preferably sixteen, biomarkers may be used to detect renal disorders. As used herein, the term “renal disorder” includes, but is not limited to glomerulonephritis, interstitial nephritis, tubular damage, vasculitis, glomerulosclerosis, diabetic nephropathy, analgesic nephropathy, and acute tubular necrosis. As used herein, the term “glomerulonephritis” refers to a disorder characterized by inflammation of the glomeruli. The term may encompass chronic glomerulonephritis, acute glomerulonephritis, primary glomerulonephritis, or secondary glomerulonephritis. As used herein, the term “diabetic nephropathy” refers to a disorder characterized by angiopathy of capillaries in the kidney glomeruli. The term encompasses Kimmelstiel-Wilson syndrome, or nodular diabetic glomerulosclerosis and intercapillary glomerulonephritis. Additionally, the present invention encompasses biomarkers that may be used to detect a disorder associated with diabetic nephropathy. As used herein, the phrase “a disorder associated with diabetic nephropathy” refers to a disorder that stems from angiopathy of capillaries in the kidney glomeruli. For instance, non-limiting examples of associated disorders may include nephritic syndrome, chronic kidney failure, and end-stage kidney disease. The devices of the present invention may also be used to detect secondary kidney damage or toxicity caused by exposure to a toxic compound including but not limited to therapeutic drugs, recreational drugs, medical imaging contrast agents, and toxins. Non-limiting examples of therapeutic drugs may include an analgesic (e.g. aspirin, acetaminophen, ibuprofen, naproxen sodium), an antibiotic (e.g. an aminoglycoside, a beta lactam (cephalosporins, penicillins, penems), rifampin, vancomycin, a sulfonamide, a fluoroquinolone, and a tetracycline), or a chemotherapy agent (e.g. Cisplatin (Platinol®), Carboplatin (Paraplatin®), Cytarabine (Cytosar-U®), Gemtuzumab ozogamicin (Mylotarg®), Gemcitabine (Gemzar®), Melphalan (Alkeran®), Ifosfamide (Ifex®), Methotrexate (Rheumatrex®), Interleukin-2 (Proleukin®), Oxaliplatin (Eloxatin®), Streptozocin (Zanosar®), Pemetrexed (Alimta®), Plicamycin (Mithracin®), and Trimetrexate (Neutrexin®). Further, the term renal disorder may include kidney damage due to kidney stones, ischemia, liver transplantation, heart transplantation, lung transplantation, or hypovolemia. Moreover, the devices of the current invention may be used to detect renal disorders including kidney damage cause by other disease states including but not limited to diabetes, hypertension, autoimmune diseases including lupus, Wegener's granulomatosis, Goodpasture syndrome, primary hyperoxaluria, kidney transplant rejection, sepsis, nephritis secondary to any infection of the kidney, rhabdomyolysis, multiple myeloma, and prostate disease.

In addition, the devices and systems of the current invention may be used to detect renal disorders including acute kidney transplant rejection or chronic allograft nephropathy. Importantly, the devices of the invention may be used to distinguish between an acute rejection reaction and a chronic allograft nephropathy. Alternatively, the devices of the present invention may be used to distinguish between a successful transplant and rejection. As used herein, the term “rejection” refers to a recipient response to a foreign antigen derived from the transplanted kidney. The phrase “acute rejection” refers to an immune related response to the foreign kidney. The response is primarily T-cell driven and originates from an HLC mismatch between the donor and recipient. The phrase “chronic allograft nephropathy” refers to a chronic inflammatory and immune response mediated reaction to a foreign kidney. Chronic allograft nephropathy may result in damage to the kidney manifested by diffuse interstitial fibrosis glomerular changes, typically membranous and sclerotic in nature, as well as intimal fibrosis of the blood vessels with tubular atrophy and loss of tubular structures.

Additionally, the present invention encompasses devices comprising biomarkers that may be used to detect a renal disorder associated with kidney transplant rejection. As used herein, the phrase “a disorder associated with kidney transplant rejection” refers to a disorder that stems from a host response to a foreign antigen derived from the transplated kidney. For instance, non-limiting examples of associated disorders may include chronic kidney failure and end-stage kidney disease.

The devices of the present invention may also be utilized to detect a renal disorder including obstructive uropathy or an associated disorder in a mammal that includes determining the presence or concentration of a combination of three or more sample analytes in a test sample containing the bodily fluid of the mammal. As used herein, the term “obstructive uropathy” refers to a structural or functional hindrance of normal urine flow. The term may encompass chronic unilateral obstructive uropathy, chronic bilateral obstructive uropathy, acute unilateral obstructive uropathy, or acute bilateral obstructive uropathy. Additionally, the present invention encompasses biomarkers that may be used to detect a disorder associated with obstructive uropathy. As used herein, the phrase “a disorder associated with obstructive uropathy” refers to a disorder that stems from a structural or functional hindrance of normal urine flow. For instance, non-limiting examples of associated disorders may include hydronephrosis and obstructive nephropathy. The measured concentrations of the combination of sample analytes is compared to the entries of a dataset in which each entry contains the minimum diagnostic concentrations of a combination of three of more analytes reflective of obstructive uropathy or an associated disorder. Other embodiments provide computer-readable media encoded with applications containing executable modules, systems that include databases and processing devices containing executable modules configured to diagnose, monitor, or determine a renal disorder in a mammal. Still other embodiments provide antibody-based devices for diagnosing, monitoring, or determining obstructive uropathy or an associated disorder in a mammal.

The biomarkers included in a multiplexed panel of the invention are analytes known in the art that may be detected in the urine, serum, plasma and other bodily fluids of mammals. As such, the analytes of the multiplexed panel may be readily extracted from the mammal in a test sample of bodily fluid. The concentrations of the analytes within the test sample may be measured using known analytical techniques such as a multiplexed antibody-based immunological assay. The combination of concentrations of the analytes in the test sample may be compared to empirically determined combinations of minimum diagnostic concentrations and combinations of diagnostic concentration ranges associated with healthy kidney function to determine whether a renal disorder is indicated in the mammal.

The analytes used as biomarkers in the multiplexed assay, methods of diagnosing, monitoring, or determining a renal disorder using measurements of the analytes, systems and applications used to analyze the multiplexed assay measurements, and antibody-based devices used to measure the analytes are described in detail below.

I. Analytes in Multiplexed Assay

One embodiment of the invention measures the concentrations of three or more, six or more, ten or more, and preferably sixteen, biomarker analytes within a test sample taken from a mammal and compares the measured analyte concentrations to minimum diagnostic concentrations to diagnose, monitor, or determine obstructive uropathy or an associated renal disorder in a mammal. In this aspect, the biomarker analytes are known in the art to occur in the urine, plasma, serum and other bodily fluids of mammals. The biomarker analytes are proteins that have known and documented associations with early renal damage in humans. As defined herein, the biomarker analytes include but are not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF. A description of each biomarker analyte is given below. In one embodiment, the biomarker analytes include alpha-1-microglobulin, beta-2-microglobulin, cystatin-C, KIM-1, THP, and TIMP-1.

(a) Alpha-1 Microglobulin (A1M)

Alpha-1 microglobulin (A1M, Swiss-Prot Accession Number P02760) is a 26 kDa glycoprotein synthesized by the liver and reabsorbed in the proximal tubules. Elevated levels of A1M in human urine are indicative of glomerulotubular dysfunction. A1M is a member of the lipocalin super family and is found in all tissues. Alpha-1-microglobulin exists in blood in both a free form and complexed with immunoglobulin A (IgA) and heme. Half of plasma A1M exists in a free form, and the remainder exists in complexes with other molecules including prothrombin, albumin, immunoglobulin A and heme. Nearly all of the free A1M in human urine is reabsorbed by the megalin receptor in proximal tubular cells, where it is then catabolized. Small amounts of A1M are excreted in the urine of healthy humans. Increased A1M concentrations in human urine may be an early indicator of renal damage, primarily in the proximal tubule.

(b) Beta-2 Microglobulin (B2M)

Beta-2 microglobulin (B2M, Swiss-Prot Accession Number P61769) is a protein found on the surfaces of all nucleated cells and is shed into the blood, particularly by tumor cells and lymphocytes. Due to its small size, B2M passes through the glomerular membrane, but normally less than 1% is excreted due to reabsorption of B2M in the proximal tubules of the kidney. Therefore, high plasma levels of B2M occur as a result of renal failure, inflammation, and neoplasms, especially those associated with B-lymphocytes.

(c) Calbindin

Calbindin (Calbindin D-28K, Swiss-Prot Accession Number P05937) is a Ca-binding protein belonging to the troponin C superfamily. It is expressed in the kidney, pancreatic islets, and brain. Calbindin is found predominantly in subpopulations of central and peripheral nervous system neurons, in certain epithelial cells involved in Ca2+ transport such as distal tubular cells and cortical collecting tubules of the kidney, and in enteric neuroendocrine cells.

(d) Clusterin

Clusterin (Swiss-Prot Accession Number P10909) is a highly conserved protein that has been identified independently by many different laboratories and named SGP2, S35-S45, apolipoprotein J, SP-40, 40, ADHC-9, gp80, GPIII, and testosterone-repressed prostate message (TRPM-2). An increase in clusterin levels has been consistently detected in apoptotic heart, brain, lung, liver, kidney, pancreas, and retinal tissue both in vivo and in vitro, establishing clusterin as a ubiquitous marker of apoptotic cell loss. However, clusterin protein has also been implicated in physiological processes that do not involve apoptosis, including the control of complement-mediated cell lysis, transport of beta-amyloid precursor protein, shuttling of aberrant beta-amyloid across the blood-brain barrier, lipid scavenging, membrane remodeling, cell aggregation, and protection from immune detection and tumor necrosis factor induced cell death.

(e) Connective Tissue Growth Factor (CTGF)

Connective tissue growth factor (CTGF, Swiss-Prot Accession Number P29279) is a 349-amino acid cysteine-rich polypeptide belonging to the CCN family. In vitro studies have shown that CTGF is mainly involved in extracellular matrix synthesis and fibrosis. Up-regulation of CTGF mRNA and increased CTGF levels have been observed in various diseases, including diabetic nephropathy and cardiomyopathy, fibrotic skin disorders, systemic sclerosis, biliary atresia, liver fibrosis and idiopathic pulmonary fibrosis, and nondiabetic acute and progressive glomerular and tubulointerstitial lesions of the kidney. A recent cross-sectional study found that urinary CTGF may act as a progression promoter in diabetic nephropathy.

(f) Creatinine

Creatinine is a metabolite of creatine phosphate in muscle tissue, and is typically produced at a relatively constant rate by the body. Creatinine is chiefly filtered out of the blood by the kidneys, though a small amount is actively secreted by the kidneys into the urine. Creatinine levels in blood and urine may be used to estimate the creatinine clearance, which is representative of the overall glomerular filtration rate (GFR), a standard measure of renal function. Variations in creatinine concentrations in the blood and urine, as well as variations in the ratio of urea to creatinine concentration in the blood, are common diagnostic measurements used to assess renal function.

(g) Cystatin C (Cyst C)

Cystatin C (Cyst C, Swiss-Prot Accession Number P01034) is a 13 kDa protein that is a potent inhibitor of the C1 family of cysteine proteases. It is the most abundant extracellular inhibitor of cysteine proteases in testis, epididymis, prostate, seminal vesicles and many other tissues. Cystatin C, which is normally expressed in vascular wall smooth muscle cells, is severely reduced in both atherosclerotic and aneurismal aortic lesions.

(h) Epidermal Growth Factor (EGF)

Epidermal growth factor (EGF, Swiss-Prot Accession Number P07522) is a small protein that functions as a potent mitogen. EGF promotes cell growth and differentiation, is essential in embryogenesis, and is important in wound healing. It is produced by many normal cell types and is made in large amounts by certain types of tumors.

(i) Glutathione S-Transferase alpha (GST-alpha)

Glutathione S-transferase alpha (GST-alpha, Swiss-Prot Accession Number P08263) belongs to a family of enzymes that utilize glutathione in reactions contributing to the transformation of a wide range of compounds, including carcinogens, therapeutic drugs, and products of oxidative stress. These enzymes play a key role in the detoxification of such substances.

(j) Glutathione S-Transferase mu (GST-mu)

Glutathione S-transferase mu (GST-mu, Swiss-Prot Accession Number PO4905) functions in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress, by conjugation with glutathione. The genes encoding the mu class of enzymes are organized in a gene cluster on chromosome 1 p13.3 and are known to be highly polymorphic. Genetic variations in GST-mu can change a mammal's susceptibility to carcinogens and toxins as well as affect the toxicity and efficacy of certain drugs. Null mutations of this class mu gene have been linked with an increase in a number of cancers.

(k) Kidney Injury Molecule-1 (KIM-1)

Kidney injury molecule-1 (KIM-1, Swiss-Prot Accession Number Q96D42) is an immunoglobulin superfamily cell-surface protein highly upregulated on the surface of injured kidney epithelial cells. It is also known as TIM-1 (T-cell immunoglobulin mucin domain-1), as it is expressed at low levels by subpopulations of activated T-cells and hepatitis A virus cellular receptor-1 (HAVCR-1). KIM-1 is increased in expression more than any other protein in the injured kidney and is localized predominantly to the apical membrane of the surviving proximal epithelial cells.

(l) Microalbumin

Albumin is the most abundant plasma protein in humans and other mammals. Albumin is essential for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. Healthy, normal kidneys typically filter out albumin from the urine. The presence of albumin in the urine may indicate damage to the kidneys. Albumin in the urine may also occur in patients with long-standing diabetes, especially type 1 diabetes. The amount of albumin eliminated in the urine has been used to differentially diagnose various renal disorders. For example, nephrotic syndrome usually results in the excretion of about 3.0 to 3.5 grams of albumin in human urine every 24 hours. Microalbuminuria, in which less than 300 mg of albumin is eliminated in the urine every 24 hours, may indicate the early stages of diabetic nephropathy.

(m) Neutrophil Gelatinase-Associated Lipocalin (NGAL)

Neutrophil gelatinase-associated lipocalin (NGAL, Swiss-Prot Accession Number P80188) forms a disulfide bond-linked heterodimer with MMP-9. It mediates an innate immune response to bacterial infection by sequestrating iron. Lipocalins interact with many different molecules such as cell surface receptors and proteases, and play a role in a variety of processes such as the progression of cancer and allergic reactions.

(n) Osteopontin (OPN)

Osteopontin (OPN, Swiss-Prot Accession Number P10451) is a cytokine involved in enhancing production of interferon-gamma and IL-12, and inhibiting the production of IL-10. OPN is essential in the pathway that leads to type I immunity. OPN appears to form an integral part of the mineralized matrix. OPN is synthesized within the kidney and has been detected in human urine at levels that may effectively inhibit calcium oxalate crystallization. Decreased concentrations of OPN have been documented in urine from patients with renal stone disease compared with normal individuals.

(o) Tamm-Horsfall Protein (THP)

Tamm-Horsfall protein (THP, Swiss-Prot Accession Number P07911), also known as uromodulin, is the most abundant protein present in the urine of healthy subjects and has been shown to decrease in individuals with kidney stones. THP is secreted by the thick ascending limb of the loop of Henley. THP is a monomeric glycoprotein of ˜85 kDa with ˜30% carbohydrate moiety that is heavily glycosylated. THP may act as a constitutive inhibitor of calcium crystallization in renal fluids.

(p) Tissue Inhibitor of Metalloproteinase-1 (TIMP-1)

Tissue inhibitor of metalloproteinase-1 (TIMP-1, Swiss-Prot Accession Number P01033) is a major regulator of extracellular matrix synthesis and degradation. A certain balance of MMPs and TIMPs is essential for tumor growth and health. Fibrosis results from an imbalance of fibrogenesis and fibrolysis, highlighting the importance of the role of the inhibition of matrix degradation role in renal disease.

(q) Trefoil Factor 3 (TFF3)

Trefoil factor 3 (TFF3, Swiss-Prot Accession Number Q07654), also known as intestinal trefoil factor, belongs to a small family of mucin-associated peptides that include TFF1, TFF2, and TFF3. TFF3 exists in a 60-amino acid monomeric form and a 118-amino acid dimeric form. Under normal conditions TFF3 is expressed by goblet cells of the intestine and the colon. TFF3 expression has also been observed in the human respiratory tract, in human goblet cells and in the human salivary gland. In addition, TFF3 has been detected in the human hypothalamus.

(r) Vascular Endothelial Growth Factor (VEGF)

Vascular endothelial growth factor (VEGF, Swiss-Prot Accession Number P15692) is an important factor in the pathophysiology of neuronal and other tumors, most likely functioning as a potent promoter of angiogenesis. VEGF may also be involved in regulating blood-brain-barrier functions under normal and pathological conditions. VEGF secreted from the stromal cells may be responsible for the endothelial cell proliferation observed in capillary hemangioblastomas, which are typically composed of abundant microvasculature and primitive angiogenic elements represented by stromal cells.

(s) Vascular Endothelial Growth Factor A (VEGF A)

Vascular endothelial growth factor A (VEGF A, Swiss-Prot Accession Number Q00731) is a growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. It induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis, and induces permeabilization of blood vessles. It is important in the pathophysiology of neuronal and other tumors, likely functioning as a potent promoter of angiogenesis. Due to its influences on vascular permeability, VEGF A may be involved in altering blood-brain-barrier functions under normal and pathological conditions. The production and secretion of VEGF by mammalian retinal pigment epithelial cells may be important in the pathogenesis of ocular neovascularization.

(t) B-lymphocyte Chemoattractant (BLC)

B-lymphocyte chemoattractant (BLC, Swiss-Prot Accession Number 043927) is also referred to as C-X-C motif chemokine 13, Small-inducible cytokine B13, B lymphocyte chemoattractant, CXC chemokine BLC, and B cell-attracting chemokine 1. BLC functions as a potent chemoattractant for B lymphocytes, but not T lymphocytes, monocytes, or neutrophils. Its specific receptor BLR1 is a G protein-coupled receptor originally isolated from Burkitt's lymphoma cells. Among cells of the hematopoietic lineages, the expression of BRL1, now designated CXCR5, is restricted to B lymphocytes and a subpopulation of T helper memory cells.

(u) Cluster of Differentiation Surface Receptors 40 (CD40)

Cluster of Differentiation Surface Receptors 40 (CD40, Swiss Prot Accession Number P25942) is also referred to TNFRSF5 (Tumor necrosis factor receptor superfamily member 5. CD40 is a member of the tumor necrosis factor-receptor superfamily of proteins. CD40 has been found to be essential in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation.

(v) Insulin-Like Growth Factor Binding Protein 2 (IGF BP2)

Insulin-like Growth Factor Binding Protein 2 (IGF BP2, Swiss Prot Accession Number P18065) functions to prolong the half-life of the insulin growth factors and have been shown to either inhibit or stimulate the growth promoting effects of the insulin growth factors on cell culture. Specifically, during development, insulin-like growth factor binding protein-2 is expressed in a number of tissues with the highest expression level found in the central nervous system. IGFBP-2 exhibits a 2-10 fold higher affinity for IGF II than for IGF I.

(w) Matrix Metalloproteinase-3 (MMP3)

Matrix Metalloproteinase-3 (MMP3, Swiss Prot Accession Number P08254) is also known as stromelysin-1 and Transin-1. MMP3 is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. MMP3 encodes an enzyme which degrades fibronectin, laminin, collagens III, IV, IX, and X, and cartilage proteoglycans. The enzyme is thought to be involved in wound repair, progression of atherosclerosis, and tumor initiation. MMP3 is part of a cluster of MMP genes which localize to chromosome 11q22.3.

(x) Peptide YY (PYY)

Peptide YY (PYY, Swiss-Prot Accession Number P10082) is also known as peptide tyrosine tyrosine and pancreatic peptide YY₃₋₃₆. Peptide YY exerts its action through neuropeptide Y receptors, inhibits gastric motility and increases water and electrolyte absorption in the colon. PYY may also suppress pancreatic secretion. It is secreted by the neuroendocrine cells in the ileum and colon in response to a meal, and has been shown to reduce appetite. PYY works by slowing the gastric emptying; hence, it increases efficiency of digestion and nutrient absorption after meal. Research has also indicated that PYY may be useful in removing aluminum accumulated in the brain.

(y) Stem Cell Factor (SCF)

Stem Cell Factor (SCF, UniProtKB/TrEMBL Q13528) is also known as kit-ligand, KL, and steel factor. SCF functions SCF plays an important role in the hematopoiesis during embryonic development. Sites where hematopoiesis takes place, such as the fetal liver and bone marrow, all express SCF. SCF may serve as guidance cues that direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which a stem cell resides), and it plays an important role in HSC maintenance. Non-lethal point mutants on the c-Kit receptor can cause anemia, decreased fertility, and decreased pigmentation. During development, the presence of the SCF also plays an important role in the localization of melanocytes, cells that produce melanin and control pigmentation. In melanogenisis, melanoblasts migrate from the neural crest to their appropriate locations in the epidermis. Melanoblasts express the Kit receptor, and it is believed that SCF guides these cells to their terminal locations. SCF also regulates survival and proliferation of fully differentiated melanocytes in adults. In spermatogenesis, c-Kit is expressed in primordial germ cells, spermatogonia, and in primordial oocytes. It is also expressed in the primordial germ cells of females. SCF is expressed along the pathways that the germ cells use to reach their terminal destination in the body. It is also expressed in the final destinations for these cells. Like for melanoblasts, this helps guide the cells to their appropriate locations in the body

(z) Tumor Necrosis Factor Receptor Type II (TNF RII)

Tumor Necrosis Factor Receptor Type II (TNF RII, Swiss-Prot Accession Number P20333) is also known as p75, p80 TNF alpha receptor, and TNFRSF1B. TNF RII is a protein that in humans is encoded by the TNFRSF1B gene. The protein encoded by this gene is a member of the Tumor necrosis factor receptor superfamily, which also contains TNFRSF1A. The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein and TNF-receptor 1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity. The function of IAPs in TNF-receptor signaling is unknown; however, c-IAP1 is thought to potentiate TNF-induced apoptosis by the ubiquitination and degradation of TNF-receptor-associated factor 2, which mediates anti-apoptotic signals. Knockout studies in mice also suggest a role of this protein in protecting neurons from apoptosis by stimulating antioxidative pathways.

(aa) AXL Oncogene

AXL (Swiss-Prot Accession Number P30530) is also known as UFO, ARK, and tyrosine-protein kinase receptor UFO. The protein encoded by AXL is a member of the receptor tyrosine kinase subfamily. Although it is similar to other receptor tyrosine kinases, the AXL protein represents a unique structure of the extracellular region that juxtaposes IgL and FNIII repeats. AXL transduces signals from the extracellular matrix into the cytoplasm by binding growth factors like vitamin K-dependent protein growth-arrest-specific gene 6. It is involved in the stimulation of cell proliferation. This receptor can also mediate cell aggregation by homophilic binding. AXL is a chronic myelogenous leukemia-associated oncogene and also associated with colon cancer and melanoma.

(bb) Eotaxin 3

Eotaxin 3 (Swiss-Prot Accession Number P51671) is also known as C-C motif chemokine 11 (CCL11), small inducible cytokine A11, and eosinophil chemotactic protein. Eotaxin 3 is a small cytokine belonging to the CC chemokine family that is also called Eotaxin-3, Macrophage inflammatory protein 4-alpha (MIP-4-alpha), Thymic stroma chemokine-1 (TSC-1), and IMAC. It is expressed by several tissues including heart, lung and ovary, and in endothelial cells that have been stimulated with the cytokine interleukin 4.[1][2] CCL26 is chemotactic for eosinophils and basophils and elicits its effects by binding to the cell surface chemokine receptor CCR3.

(cc) Fatty Acid Binding Protein (FABP)

Fatty Acid Binding Protein (FABP, Swiss-Prot Accession Number Q01469) is also known as epidermal-type fatty acid binding protein, and fatty-acid binding protein 5. This gene encodes the fatty acid binding protein found in epidermal cells, and was first identified as being upregulated in psoriasis tissue. Fatty acid binding proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It is thought that FABPs roles include fatty acid uptake, transport, and metabolism.

(dd) Basic Fibroblast Growth Factor (FGF basic)

Basic Fibroblast Growth Factor (FGF basic, Swiss-Prot Accession NumberP09038) is also known as heparin-binding growth factor. In normal tissue, basic fibroblast growth factor is present in basement membranes and in the subendothelial extracellular matrix of blood vessels. It stays membrane-bound as long as there is no signal peptide. It has been hypothesized that, during both wound healing of normal tissues and tumor development, the action of heparan sulfate-degrading enzymes activates FGF basic, thus mediating the formation of new blood vessels. Additionally, FGF basic is a critical component of human embryonic stem cell culture medium; the growth factor is necessary for the cells to remain in an undifferentiated state, although the mechanisms by which it does this are poorly defined. It has been demonstrated to induce gremlin expression which in turn is known to inhibit the induction of differentiation by bone morphogenetic proteins. It is necessary in mouse-feeder cell dependent culture systems, as well as in feeder and serum-free culture systems.

(ee) Myoglobin

Myoglobin (Swiss-Prot Accession Number P02144) is released from damaged muscle tissue (rhabdomyolysis), which has very high concentrations of myoglobin. The released myoglobin is filtered by the kidneys but is toxic to the renal tubular epithelium and so may cause acute renal failure. Myoglobin is a sensitive marker for muscle injury, making it a potential marker for heart attack in patients with chest pain.

(ff) Resistin (RETN)

Resistin (RETN, UniProtKB/TrEMBL Q76B53) is theorized to participate in the inflammatory response. Resistin has also been shown to increase transcriptional events leading to an increased expression of several pro-inflammatory cytokines including (but not limited to) interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) in an NF-KB-mediated fashion. It has also been demonstrated that resistin upregulates intracellular adhesion molecule-1 (ICAM1) vascular cell-adhesion molecule-1 (VCAM1) and CCL2, all of which are occupied in chemotactic pathways involved in leukocyte recruitment to sites of infection. Resistin itself can be upregulated by interleukins and also by microbial antigens such as lipopolysaccharide, which are recognized by leukocytes. Taken together, because resistin is reputed to contribute to insulin resistance, results such as those mentioned suggest that resistin may be a link in the well-known association between inflammation and insulin resistance. In fact, recent data have shown positive correlations between obesity, insulin resistance, and chronic inflammation which is believed to be directed in part by resistin signaling.

(gg) Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 3 (TRAIL R3)

TRAIL R3 (Swiss-Prot Accession Number P83626 (mouse)) is also known as tumor necrosis factor-related apoptosis-inducing ligand receptor 3, and tumor necrosis factor receptor mouse homolog. TRAIL R3 is a decoy receptor for TRAIL, a member of the tumor necrosis factor family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and thus preventing its binding to proapoptotic TRAIL receptors.

(hh) Endothelin 1 (ET1)

Endothelin 1 (ET1, UniProtKB/TrEMBL Q6FH53) is also known as EDN1 and EDN1 protein. Endothelin 1 is a protein that constricts blood vessels and raises blood pressure. It is normally kept in balance by other mechanisms, but when over-expressed, it contributes to high blood pressure (hypertension) and heart disease. Endothelin 1 peptides and receptors are implicated in the pathogenesis of a number of disease states, including cancer and heart disease.

(ii) Neuronal Cell Adhesion Molecule (NrCAM)

Neuronal Cell Adhesion Molecule (NrCAM, UniProtKB/TrEMBL Q14CA1) encodes a neuronal cell adhesion molecule with multiple immunoglobulin-like C2-type domains and fibronectin type-III domains. This ankyrin-binding protein is involved in neuron-neuron adhesion and promotes directional signaling during axonal cone growth. This gene is also expressed in non-neural tissues and may play a general role in cell-cell communication via signaling from its intracellular domain to the actin cytoskeleton during directional cell migration. Allelic variants of this gene have been associated with autism and addiction vulnerability.

(jj) Tenascin C (TN-C)

Tenascin C (TN-C, UniProt/TrEMBL Q99857) has anti-adhesive properties, causing cells in tissue culture to become rounded after it is added to the medium. One mechanism to explain this may come from its ability to bind to the extracellular matrix glycoprotein fibronectin and block fibronectin's interactions with specific syndecans. The expression of tenascin-C in the stroma of certain tumors is associated with a poor prognosis.

(kk) Vascular Cell Adhesion Molecule 1 (VCAM1)

Vascular Cell Adhesion Molecule 1 (VCAM1, Swiss-Prot Accession Number P19320) is also known as vascular cell adhesion protein 1. VCAM1 mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis. Upregulation of VCAM-1 in endothelial cells by cytokines occurs as a result of increased gene transcription (e.g., in response to Tumor necrosis factor-alpha (TNF-α) and Interleukin-1 (IL-1)) and through stabilization of Messenger RNA (mRNA) (e.g., Interleukin-4 (IL-4)). The promoter region of the VCAM-1 gene contains functional tandem NF-κB (nuclear factor-kappa B) sites. The sustained expression of VCAM-1 lasts over 24 hours. Primarily, the VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 or α4β1) of the β1 subfamily of integrins, and for integrin α4β7. VCAM-1 expression has also been observed in other cell types (e.g., smooth muscle cells). It has also been shown to interact with EZR and Moesin. Certain melanoma cells can use VCAM-1 to adhere to the endothelium, and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites.

(ll) Cortisol

Cortisol (Swiss-Prot Accession Number P08185) is also known as corticosteroid-binding globulin, transcortin, and Serpin A6. Cortisol is a steroid hormone or glucocorticoid produced by the adrenal gland. It is released in response to stress, and to a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis, suppress the immune system, and aid in fat, protein and carbohydrate metabolism. It also decreases bone formation. In addition, cortisol can weaken the activity of the immune system. Cortisol prevents proliferation of T-cells by rendering the interleukin-2 producer T-cells unresponsive to interleukin-1 (IL-1), and unable to produce the T-cell growth factor. Cortisol also has a negative feedback effect on interleukin-1. IL-1 must be especially useful in combating some diseases; however, endotoxin bacteria have gained an advantage by forcing the hypothalamus to increase cortisol levels via forcing secretion of CRH hormone, thus antagonizing IL-1 in this case. The suppressor cells are not affected by GRMF, so that the effective set point for the immune cells may be even higher than the set point for physiological processes. It reflects leukocyte redistribution to lymph nodes, bone marrow, and skin.

II. Combinations of Analytes Measured by Multiplexed Assay

The device for diagnosing, monitoring, or determining a renal disorder involves determining the presence or concentrations of a combination of sample analytes in a test sample. The combinations of sample analytes, as defined herein, are any group of three or more analytes selected from the biomarker analytes, including but not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol. In one embodiment, the combination of analytes may be selected to provide a group of analytes associated with renal disorder in a mammal.

In one embodiment, the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes. In other embodiments, the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, or all sixteen of the sixteen biomarker analytes. In another embodiment, the combination of sample analytes may comprise a combination listed in Table A.

TABLE A alpha-1 microglobulin beta-2 microglobulin calbindin alpha-1 microglobulin beta-2 microglobulin clusterin alpha-1 microglobulin beta-2 microglobulin CTGF alpha-1 microglobulin beta-2 microglobulin creatinine alpha-1 microglobulin beta-2 microglobulin cystatin C alpha-1 microglobulin beta-2 microglobulin GST-alpha alpha-1 microglobulin beta-2 microglobulin KIM-1 alpha-1 microglobulin beta-2 microglobulin microalbumin alpha-1 microglobulin beta-2 microglobulin NGAL alpha-1 microglobulin beta-2 microglobulin osteopontin alpha-1 microglobulin beta-2 microglobulin THP alpha-1 microglobulin beta-2 microglobulin TIMP-1 alpha-1 microglobulin beta-2 microglobulin TFF-3 alpha-1 microglobulin beta-2 microglobulin VEGF alpha-1 microglobulin calbindin clusterin alpha-1 microglobulin calbindin CTGF alpha-1 microglobulin calbindin creatinine alpha-1 microglobulin calbindin cystatin C alpha-1 microglobulin calbindin GST-alpha alpha-1 microglobulin calbindin KIM-1 alpha-1 microglobulin calbindin microalbumin alpha-1 microglobulin calbindin NGAL alpha-1 microglobulin calbindin osteopontin alpha-1 microglobulin calbindin THP alpha-1 microglobulin calbindin TIMP-1 alpha-1 microglobulin calbindin TFF-3 alpha-1 microglobulin calbindin VEGF alpha-1 microglobulin clusterin CTGF alpha-1 microglobulin clusterin creatinine alpha-1 microglobulin clusterin cystatin C alpha-1 microglobulin clusterin GST-alpha alpha-1 microglobulin clusterin KIM-1 alpha-1 microglobulin clusterin microalbumin alpha-1 microglobulin clusterin NGAL alpha-1 microglobulin clusterin osteopontin alpha-1 microglobulin clusterin THP alpha-1 microglobulin clusterin TIMP-1 alpha-1 microglobulin clusterin TFF-3 alpha-1 microglobulin clusterin VEGF alpha-1 microglobulin CTGF creatinine alpha-1 microglobulin CTGF cystatin C alpha-1 microglobulin CTGF GST-alpha alpha-1 microglobulin CTGF KIM-1 alpha-1 microglobulin CTGF microalbumin alpha-1 microglobulin CTGF NGAL alpha-1 microglobulin CTGF osteopontin alpha-1 microglobulin CTGF THP alpha-1 microglobulin CTGF TIMP-1 alpha-1 microglobulin CTGF TFF-3 alpha-1 microglobulin CTGF VEGF alpha-1 microglobulin creatinine cystatin C alpha-1 microglobulin creatinine GST-alpha alpha-1 microglobulin creatinine KIM-1 alpha-1 microglobulin creatinine microalbumin alpha-1 microglobulin creatinine NGAL alpha-1 microglobulin creatinine osteopontin alpha-1 microglobulin creatinine THP alpha-1 microglobulin creatinine TIMP-1 alpha-1 microglobulin creatinine TFF-3 alpha-1 microglobulin creatinine VEGF alpha-1 microglobulin cystatin C GST-alpha alpha-1 microglobulin cystatin C KIM-1 alpha-1 microglobulin cystatin C microalbumin alpha-1 microglobulin cystatin C NGAL alpha-1 microglobulin cystatin C osteopontin alpha-1 microglobulin cystatin C THP alpha-1 microglobulin cystatin C TIMP-1 alpha-1 microglobulin cystatin C TFF-3 alpha-1 microglobulin cystatin C VEGF alpha-1 microglobulin GST-alpha KIM-1 alpha-1 microglobulin GST-alpha microalbumin alpha-1 microglobulin GST-alpha NGAL alpha-1 microglobulin GST-alpha osteopontin alpha-1 microglobulin GST-alpha THP alpha-1 microglobulin GST-alpha TIMP-1 alpha-1 microglobulin GST-alpha TFF-3 alpha-1 microglobulin GST-alpha VEGF alpha-1 microglobulin KIM-1 microalbumin alpha-1 microglobulin KIM-1 NGAL alpha-1 microglobulin KIM-1 osteopontin alpha-1 microglobulin KIM-1 THP alpha-1 microglobulin KIM-1 TIMP-1 alpha-1 microglobulin KIM-1 TFF-3 alpha-1 microglobulin KIM-1 VEGF alpha-1 microglobulin microalbumin NGAL alpha-1 microglobulin microalbumin osteopontin alpha-1 microglobulin microalbumin THP alpha-1 microglobulin microalbumin TIMP-1 alpha-1 microglobulin microalbumin TFF-3 alpha-1 microglobulin microalbumin VEGF alpha-1 microglobulin NGAL osteopontin alpha-1 microglobulin NGAL THP alpha-1 microglobulin NGAL TIMP-1 alpha-1 microglobulin NGAL TFF-3 alpha-1 microglobulin NGAL VEGF alpha-1 microglobulin osteopontin THP alpha-1 microglobulin osteopontin TIMP-1 alpha-1 microglobulin osteopontin TFF-3 alpha-1 microglobulin osteopontin VEGF alpha-1 microglobulin THP TIMP-1 alpha-1 microglobulin THP TFF-3 alpha-1 microglobulin THP VEGF alpha-1 microglobulin TIMP-1 TFF-3 alpha-1 microglobulin TIMP-1 VEGF alpha-1 microglobulin TFF-3 VEGF beta-2 microglobulin calbindin clusterin beta-2 microglobulin calbindin CTGF beta-2 microglobulin calbindin creatinine beta-2 microglobulin calbindin cystatin C beta-2 microglobulin calbindin GST-alpha beta-2 microglobulin calbindin KIM-1 beta-2 microglobulin calbindin microalbumin beta-2 microglobulin calbindin NGAL beta-2 microglobulin calbindin osteopontin beta-2 microglobulin calbindin THP beta-2 microglobulin calbindin TIMP-1 beta-2 microglobulin calbindin TFF-3 beta-2 microglobulin calbindin VEGF beta-2 microglobulin clusterin CTGF beta-2 microglobulin clusterin creatinine beta-2 microglobulin clusterin cystatin C beta-2 microglobulin clusterin GST-alpha beta-2 microglobulin clusterin KIM-1 beta-2 microglobulin clusterin microalbumin beta-2 microglobulin clusterin NGAL beta-2 microglobulin clusterin osteopontin beta-2 microglobulin clusterin THP beta-2 microglobulin clusterin TIMP-1 beta-2 microglobulin clusterin TFF-3 beta-2 microglobulin clusterin VEGF beta-2 microglobulin CTGF creatinine beta-2 microglobulin CTGF cystatin C beta-2 microglobulin CTGF GST-alpha beta-2 microglobulin CTGF KIM-1 beta-2 microglobulin CTGF microalbumin beta-2 microglobulin CTGF NGAL beta-2 microglobulin CTGF osteopontin beta-2 microglobulin CTGF THP beta-2 microglobulin CTGF TIMP-1 beta-2 microglobulin CTGF TFF-3 beta-2 microglobulin CTGF VEGF beta-2 microglobulin creatinine cystatin C beta-2 microglobulin creatinine GST-alpha beta-2 microglobulin creatinine KIM-1 beta-2 microglobulin creatinine microalbumin beta-2 microglobulin creatinine NGAL beta-2 microglobulin creatinine osteopontin beta-2 microglobulin creatinine THP beta-2 microglobulin creatinine TIMP-1 beta-2 microglobulin creatinine TFF-3 beta-2 microglobulin creatinine VEGF beta-2 microglobulin cystatin C GST-alpha beta-2 microglobulin cystatin C KIM-1 beta-2 microglobulin cystatin C microalbumin beta-2 microglobulin cystatin C NGAL beta-2 microglobulin cystatin C osteopontin beta-2 microglobulin cystatin C THP beta-2 microglobulin cystatin C TIMP-1 beta-2 microglobulin cystatin C TFF-3 beta-2 microglobulin cystatin C VEGF beta-2 microglobulin GST-alpha KIM-1 beta-2 microglobulin GST-alpha microalbumin beta-2 microglobulin GST-alpha NGAL beta-2 microglobulin GST-alpha osteopontin beta-2 microglobulin GST-alpha THP beta-2 microglobulin GST-alpha TIMP-1 beta-2 microglobulin GST-alpha TFF-3 beta-2 microglobulin GST-alpha VEGF beta-2 microglobulin KIM-1 microalbumin beta-2 microglobulin KIM-1 NGAL beta-2 microglobulin KIM-1 osteopontin beta-2 microglobulin KIM-1 THP beta-2 microglobulin KIM-1 TIMP-1 beta-2 microglobulin KIM-1 TFF-3 beta-2 microglobulin KIM-1 VEGF beta-2 microglobulin microalbumin NGAL beta-2 microglobulin microalbumin osteopontin beta-2 microglobulin microalbumin THP beta-2 microglobulin microalbumin TIMP-1 beta-2 microglobulin microalbumin TFF-3 beta-2 microglobulin microalbumin VEGF beta-2 microglobulin NGAL osteopontin beta-2 microglobulin NGAL THP beta-2 microglobulin NGAL TIMP-1 beta-2 microglobulin NGAL TFF-3 beta-2 microglobulin NGAL VEGF beta-2 microglobulin osteopontin THP beta-2 microglobulin osteopontin TIMP-1 beta-2 microglobulin osteopontin TFF-3 beta-2 microglobulin osteopontin VEGF beta-2 microglobulin THP TIMP-1 beta-2 microglobulin THP TFF-3 beta-2 microglobulin THP VEGF beta-2 microglobulin TIMP-1 TFF-3 beta-2 microglobulin TIMP-2 VEGF beta-2 microglobulin TFF-3 VEGF calbindin clusterin CTGF calbindin clusterin creatinine calbindin clusterin cystatin C calbindin clusterin GST-alpha calbindin clusterin KIM-1 calbindin clusterin microalbumin calbindin clusterin NGAL calbindin clusterin osteopontin calbindin clusterin THP calbindin clusterin TIMP-1 calbindin clusterin TFF-3 calbindin clusterin VEGF calbindin CTGF creatinine calbindin CTGF cystatin C calbindin CTGF GST-alpha calbindin CTGF KIM-1 calbindin CTGF microalbumin calbindin CTGF NGAL calbindin CTGF osteopontin calbindin CTGF THP calbindin CTGF TIMP-1 calbindin CTGF TFF-3 calbindin CTGF VEGF calbindin creatinine cystatin C calbindin creatinine GST-alpha calbindin creatinine KIM-1 calbindin creatinine microalbumin calbindin creatinine NGAL calbindin creatinine osteopontin calbindin creatinine THP calbindin creatinine TIMP-1 calbindin creatinine TFF-3 calbindin creatinine VEGF calbindin cystatin C GST-alpha calbindin cystatin C KIM-1 calbindin cystatin C microalbumin calbindin cystatin C NGAL calbindin cystatin C osteopontin calbindin cystatin C THP calbindin cystatin C TIMP-1 calbindin cystatin C TFF-3 calbindin cystatin C VEGF calbindin GST-alpha KIM-1 calbindin GST-alpha microalbumin calbindin GST-alpha NGAL calbindin GST-alpha osteopontin calbindin GST-alpha THP calbindin GST-alpha TIMP-1 calbindin GST-alpha TFF-3 calbindin GST-alpha VEGF calbindin KIM-1 microalbumin calbindin KIM-1 NGAL calbindin KIM-1 osteopontin calbindin KIM-1 THP calbindin KIM-1 TIMP-1 calbindin KIM-1 TFF-3 calbindin KIM-1 VEGF calbindin microalbumin NGAL calbindin microalbumin osteopontin calbindin microalbumin THP calbindin microalbumin TIMP-1 calbindin microalbumin TFF-3 calbindin microalbumin VEGF calbindin NGAL osteopontin calbindin NGAL THP calbindin NGAL TIMP-1 calbindin NGAL TFF-3 calbindin NGAL VEGF calbindin osteopontin THP calbindin osteopontin TIMP-1 calbindin osteopontin TFF-3 calbindin osteopontin VEGF calbindin THP TIMP-1 calbindin THP TFF-3 calbindin THP VEGF calbindin TIMP-1 TFF-3 calbindin TIMP-1 VEGF calbindin TFF-3 VEGF clusterin CTGF creatinine clusterin CTGF cystatin C clusterin CTGF GST-alpha clusterin CTGF KIM-1 clusterin CTGF microalbumin clusterin CTGF NGAL clusterin CTGF osteopontin clusterin CTGF THP clusterin CTGF TIMP-1 clusterin CTGF TFF-3 clusterin CTGF VEGF clusterin creatinine cystatin C clusterin creatinine GST-alpha clusterin creatinine KIM-1 clusterin creatinine microalbumin clusterin creatinine NGAL clusterin creatinine osteopontin clusterin creatinine THP clusterin creatinine TIMP-1 clusterin creatinine TFF-3 clusterin creatinine VEGF clusterin cystatin C GST-alpha clusterin cystatin C KIM-1 clusterin cystatin C microalbumin clusterin cystatin C NGAL clusterin cystatin C osteopontin clusterin cystatin C THP clusterin cystatin C TIMP-1 clusterin cystatin C TFF-3 clusterin cystatin C VEGF clusterin GST-alpha KIM-1 clusterin GST-alpha microalbumin clusterin GST-alpha NGAL clusterin GST-alpha osteopontin clusterin GST-alpha THP clusterin GST-alpha TIMP-1 clusterin GST-alpha TFF-3 clusterin GST-alpha VEGF clusterin KIM-1 microalbumin clusterin KIM-1 NGAL clusterin KIM-1 osteopontin clusterin KIM-1 THP clusterin KIM-1 TIMP-1 clusterin KIM-1 TFF-3 clusterin KIM-1 VEGF clusterin microalbumin NGAL clusterin microalbumin osteopontin clusterin microalbumin THP clusterin microalbumin TIMP-1 clusterin microalbumin TFF-3 clusterin microalbumin VEGF clusterin NGAL osteopontin clusterin NGAL THP clusterin NGAL TIMP-1 clusterin NGAL TFF-3 clusterin NGAL VEGF clusterin osteopontin THP clusterin osteopontin TIMP-1 clusterin osteopontin TFF-3 clusterin osteopontin VEGF clusterin THP TIMP-1 clusterin THP TFF-3 clusterin THP VEGF clusterin TIMP-1 TFF-3 clusterin TIMP-1 VEGF clusterin TFF-3 VEGF CTGF creatinine cystatin C CTGF creatinine GST-alpha CTGF creatinine KIM-1 CTGF creatinine microalbumin CTGF creatinine NGAL CTGF creatinine osteopontin CTGF creatinine THP CTGF creatinine TIMP-1 CTGF creatinine TFF-3 CTGF creatinine VEGF CTGF cystatin C GST-alpha CTGF cystatin C KIM-1 CTGF cystatin C microalbumin CTGF cystatin C NGAL CTGF cystatin C osteopontin CTGF cystatin C THP CTGF cystatin C TIMP-1 CTGF cystatin C TFF-3 CTGF cystatin C VEGF CTGF GST-alpha KIM-1 CTGF GST-alpha microalbumin CTGF GST-alpha NGAL CTGF GST-alpha osteopontin CTGF GST-alpha THP CTGF GST-alpha TIMP-1 CTGF GST-alpha TFF-3 CTGF GST-alpha VEGF CTGF KIM-1 microalbumin CTGF KIM-1 NGAL CTGF KIM-1 osteopontin CTGF KIM-1 THP CTGF KIM-1 TIMP-1 CTGF KIM-1 TFF-3 CTGF KIM-1 VEGF CTGF microalbumin NGAL CTGF microalbumin osteopontin CTGF microalbumin THP CTGF microalbumin TIMP-1 CTGF microalbumin TFF-3 CTGF microalbumin VEGF CTGF NGAL osteopontin CTGF NGAL THP CTGF NGAL TIMP-1 CTGF NGAL TFF-3 CTGF NGAL VEGF CTGF osteopontin THP CTGF osteopontin TIMP-1 CTGF osteopontin TFF-3 CTGF osteopontin VEGF CTGF THP TIMP-1 CTGF THP TFF-3 CTGF THP VEGF CTGF TIMP-1 TFF-3 CTGF TIMP-1 VEGF CTGF TFF-3 VEGF creatinine cystatin C GST-alpha creatinine cystatin C KIM-1 creatinine cystatin C microalbumin creatinine cystatin C NGAL creatinine cystatin C osteopontin creatinine cystatin C THP creatinine cystatin C TIMP-1 creatinine cystatin C TFF-3 creatinine cystatin C VEGF creatinine GST-alpha KIM-1 creatinine GST-alpha microalbumin creatinine GST-alpha NGAL creatinine GST-alpha osteopontin creatinine GST-alpha THP creatinine GST-alpha TIMP-1 creatinine GST-alpha TFF-3 creatinine GST-alpha VEGF creatinine KIM-1 microalbumin creatinine KIM-1 NGAL creatinine KIM-1 osteopontin creatinine KIM-1 THP creatinine KIM-1 TIMP-1 creatinine KIM-1 TFF-3 creatinine KIM-1 VEGF creatinine microalbumin NGAL creatinine microalbumin osteopontin creatinine microalbumin THP creatinine microalbumin TIMP-1 creatinine microalbumin TFF-3 creatinine microalbumin VEGF creatinine NGAL osteopontin creatinine NGAL THP creatinine NGAL TIMP-1 creatinine NGAL TFF-3 creatinine NGAL VEGF creatinine osteopontin THP creatinine osteopontin TIMP-1 creatinine osteopontin TFF-3 creatinine osteopontin VEGF creatinine THP TIMP-1 creatinine THP TFF-3 creatinine THP VEGF creatinine TIMP-1 TFF-3 creatinine TIMP-1 VEGF creatinine TFF-3 VEGF cystatin C GST-alpha KIM-1 cystatin C GST-alpha microalbumin cystatin C GST-alpha NGAL cystatin C GST-alpha osteopontin cystatin C GST-alpha THP cystatin C GST-alpha TIMP-1 cystatin C GST-alpha TFF-3 cystatin C GST-alpha VEGF cystatin C KIM-1 microalbumin cystatin C KIM-1 NGAL cystatin C KIM-1 osteopontin cystatin C KIM-1 THP cystatin C KIM-1 TIMP-1 cystatin C KIM-1 TFF-3 cystatin C KIM-1 VEGF cystatin C microalbumin NGAL cystatin C microalbumin osteopontin cystatin C microalbumin THP cystatin C microalbumin TIMP-1 cystatin C microalbumin TFF-3 cystatin C microalbumin VEGF cystatin C NGAL osteopontin cystatin C NGAL THP cystatin C NGAL TIMP-1 cystatin C NGAL TFF-3 cystatin C NGAL VEGF cystatin C osteopontin THP cystatin C osteopontin TIMP-1 cystatin C osteopontin TFF-3 cystatin C osteopontin VEGF cystatin C THP TIMP-1 cystatin C THP TFF-3 cystatin C THP VEGF cystatin C TIMP-1 TFF-3 cystatin C TIMP-1 VEGF cystatin C TFF-3 VEGF GST-alpha KIM-1 microalbumin GST-alpha KIM-1 NGAL GST-alpha KIM-1 osteopontin GST-alpha KIM-1 THP GST-alpha KIM-1 TIMP-1 GST-alpha KIM-1 TFF-3 GST-alpha KIM-1 VEGF GST-alpha microalbumin NGAL GST-alpha microalbumin osteopontin GST-alpha microalbumin THP GST-alpha microalbumin TIMP-1 GST-alpha microalbumin TFF-3 GST-alpha microalbumin VEGF GST-alpha NGAL osteopontin GST-alpha NGAL THP GST-alpha NGAL TIMP-1 GST-alpha NGAL TFF-3 GST-alpha NGAL VEGF GST-alpha osteopontin THP GST-alpha osteopontin TIMP-1 GST-alpha osteopontin TFF-3 GST-alpha osteopontin VEGF GST-alpha THP TIMP-1 GST-alpha THP TFF-3 GST-alpha THP VEGF GST-alpha TIMP-1 TFF-3 GST-alpha TIMP-1 VEGF GST-alpha TFF-3 VEGF KIM-1 microalbumin NGAL KIM-1 microalbumin osteopontin KIM-1 microalbumin THP KIM-1 microalbumin TIMP-1 KIM-1 microalbumin TFF-3 KIM-1 microalbumin VEGF KIM-1 NGAL osteopontin KIM-1 NGAL THP KIM-1 NGAL TIMP-1 KIM-1 NGAL TFF-3 KIM-1 NGAL VEGF KIM-1 osteopontin THP KIM-1 osteopontin TIMP-1 KIM-1 osteopontin TFF-3 KIM-1 osteopontin VEGF KIM-1 THP TIMP-1 KIM-1 THP TFF-3 KIM-1 THP VEGF KIM-1 TIMP-1 TFF-3 KIM-1 TIMP-1 VEGF KIM-1 TFF-3 VEGF microalbumin NGAL osteopontin microalbumin NGAL THP microalbumin NGAL TIMP-1 microalbumin NGAL TFF-3 microalbumin NGAL VEGF microalbumin osteopontin THP microalbumin osteopontin TIMP-1 microalbumin osteopontin TFF-3 microalbumin osteopontin VEGF microalbumin THP TIMP-1 microalbumin THP TFF-3 microalbumin THP VEGF microalbumin TIMP-1 TFF-3 microalbumin TIMP-1 VEGF microalbumin TFF-3 VEGF NGAL osteopontin THP NGAL osteopontin TIMP-1 NGAL osteopontin TFF-3 NGAL osteopontin VEGF NGAL THP TIMP-1 NGAL THP TFF-3 NGAL THP VEGF NGAL TIMP-1 TFF-3 NGAL TIMP-1 VEGF NGAL TFF-3 VEGF osteopontin THP TIMP-1 osteopontin THP TFF-3 osteopontin THP VEGF osteopontin TIMP-1 TFF-3 osteopontin TIMP-1 VEGF osteopontin TFF-3 VEGF THP TIMP-1 TFF-3 THP TIMP-1 VEGF THP TFF-3 VEGF TIMP-1 TFF-3 VEGF

In one exemplary embodiment, the combination of sample analytes may include creatinine, KIM-1, and THP. In another exemplary embodiment, the combination of sample analytes may include microalbumin, creatinine, and KIM-1. In yet another exemplary embodiment, the combination of sample analytes may include creatinine, THP, and A1M. In still another exemplary embodiment, the combination of sample analytes may include microalbumin, TIMP-1, and osteopontin.

In still another embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of obstructive uropathy. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In an additional embodiment, the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy include three or more biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy includes six biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin.

In yet another embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of glomerulonephritis. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In an additional embodiment, the devices and systems to diagnose, monitor or determine the presence of glomerulonephritis include three or more biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of glomerulonephropathy includes six biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.

In an additional embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of kidney damage or toxicity. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In anotherembodiment, the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include three or more biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include six biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.

In a further embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of diabetic nephropathy. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In another embodiment, the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include three or more biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include six biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.

In another embodiment, the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes discussed previously to diagnose kidney transplant rejection or other associated disease as discussed previously. In other embodiments, the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, any sixteen, any seventeen, any eighteen, or any nineteen biomarker analytes. In another embodiment, the combination of sample analytes may comprise a combination listed in Table B.

TABLE B BLC CD40 IGF BP2 BLC CD40 MMP3 BLC CD40 peptide YY BLC CD40 stem cell factor BLC CD40 TNF RII BLC CD40 AXL BLC CD40 Eotaxin 3 BLC CD40 FABP BLC CD40 FGF basic BLC CD40 myoglobin BLC CD40 resistin BLC CD40 TRAIL R3 BLC CD40 endothilin 1 BLC CD40 NrCAM BLC CD40 Tenascin C BLC CD40 VCAM1 BLC CD40 cortisol BLC IGF BP2 MMP3 BLC IGF BP2 peptide YY BLC IGF BP2 stem cell factor BLC IGF BP2 TNF RII BLC IGF BP2 AXL BLC IGF BP2 Eotaxin 3 BLC IGF BP2 FABP BLC IGF BP2 FGF basic BLC IGF BP2 myoglobin BLC IGF BP2 resistin BLC IGF BP2 TRAIL R3 BLC IGF BP2 endothilin 1 BLC IGF BP2 NrCAM BLC IGF BP2 Tenascin C BLC IGF BP2 VCAM1 BLC IGF BP2 cortisol BLC MMP3 peptide YY BLC MMP3 stem cell factor BLC MMP3 TNF RII BLC MMP3 AXL BLC MMP3 Eotaxin 3 BLC MMP3 FABP BLC MMP3 FGF basic BLC MMP3 myoglobin BLC MMP3 resistin BLC MMP3 TRAIL R3 BLC MMP3 endothilin 1 BLC MMP3 NrCAM BLC MMP3 Tenascin C BLC MMP3 VCAM1 BLC MMP3 cortisol BLC peptide YY stem cell factor BLC peptide YY TNF RII BLC peptide YY AXL BLC peptide YY Eotaxin 3 BLC peptide YY FABP BLC peptide YY FGF basic BLC peptide YY myoglobin BLC peptide YY resistin BLC peptide YY TRAIL R3 BLC peptide YY endothilin 1 BLC peptide YY NrCAM BLC peptide YY Tenascin C BLC peptide YY VCAM1 BLC peptide YY cortisol BLC stem cell factor TNF RII BLC stem cell factor AXL BLC stem cell factor Eotaxin 3 BLC stem cell factor FABP BLC stem cell factor FGF basic BLC stem cell factor myoglobin BLC stem cell factor resistin BLC stem cell factor TRAIL R3 BLC stem cell factor endothilin 1 BLC stem cell factor NrCAM BLC stem cell factor Tenascin C BLC stem cell factor VCAM1 BLC stem cell factor cortisol BLC TNF RII AXL BLC TNF RII Eotaxin 3 BLC TNF RII FABP BLC TNF RII FGF basic BLC TNF RII myoglobin BLC TNF RII resistin BLC TNF RII TRAIL R3 BLC TNF RII endothilin 1 BLC TNF RII NrCAM BLC TNF RII Tenascin C BLC TNF RII VCAM1 BLC TNF RII cortisol BLC AXL Eotaxin 3 BLC AXL FABP BLC AXL FGF basic BLC AXL myoglobin BLC AXL resistin BLC AXL TRAIL R3 BLC AXL endothilin 1 BLC AXL NrCAM BLC AXL Tenascin C BLC AXL VCAM1 BLC AXL cortisol BLC Eotaxin 3 FABP BLC Eotaxin 3 FGF basic BLC Eotaxin 3 myoglobin BLC Eotaxin 3 resistin BLC Eotaxin 3 TRAIL R3 BLC Eotaxin 3 endothilin 1 BLC Eotaxin 3 NrCAM BLC Eotaxin 3 Tenascin C BLC Eotaxin 3 VCAM1 BLC Eotaxin 3 cortisol BLC FABP FGF basic BLC FABP myoglobin BLC FABP resistin BLC FABP TRAIL R3 BLC FABP endothilin 1 BLC FABP NrCAM BLC FABP Tenascin C BLC FABP VCAM1 BLC FABP cortisol BLC FGF basic myoglobin BLC FGF basic resistin BLC FGF basic TRAIL R3 BLC FGF basic endothilin 1 BLC FGF basic NrCAM BLC FGF basic Tenascin C BLC FGF basic VCAM1 BLC FGF basic cortisol BLC myoglobin resistin BLC myoglobin TRAIL R3 BLC myoglobin endothilin 1 BLC myoglobin NrCAM BLC myoglobin Tenascin C BLC myoglobin VCAM1 BLC myoglobin cortisol BLC resistin TRAIL R3 BLC resistin endothilin 1 BLC resistin NrCAM BLC resistin Tenascin C BLC resistin VCAM1 BLC resistin cortisol BLC TRAIL R3 endothilin 1 BLC TRAIL R3 NrCAM BLC TRAIL R3 Tenascin C BLC TRAIL R3 VCAM1 BLC TRAIL R3 cortisol BLC endothilin 1 NrCAM BLC endothilin 1 Tenascin C BLC endothilin 1 VCAM1 BLC endothilin 1 cortisol BLC NrCAM Tenascin C BLC NrCAM VCAM1 BLC NrCAM cortisol BLC Tenascin C VCAM1 BLC Tenascin C cortisol BLC VCAM1 cortisol CD40 IGF BP2 MMP3 CD40 IGF BP2 peptide YY CD40 IGF BP2 stem cell factor CD40 IGF BP2 TNF RII CD40 IGF BP2 AXL CD40 IGF BP2 Eotaxin 3 CD40 IGF BP2 FABP CD40 IGF BP2 FGF basic CD40 IGF BP2 myoglobin CD40 IGF BP2 resistin CD40 IGF BP2 TRAIL R3 CD40 IGF BP2 endothilin 1 CD40 IGF BP2 NrCAM CD40 IGF BP2 Tenascin C CD40 IGF BP2 VCAM1 CD40 IGF BP2 cortisol CD40 MMP3 peptide YY CD40 MMP3 stem cell factor CD40 MMP3 TNF RII CD40 MMP3 AXL CD40 MMP3 Eotaxin 3 CD40 MMP3 FABP CD40 MMP3 FGF basic CD40 MMP3 myoglobin CD40 MMP3 resistin CD40 MMP3 TRAIL R3 CD40 MMP3 endothilin 1 CD40 MMP3 NrCAM CD40 MMP3 Tenascin C CD40 MMP3 VCAM1 CD40 MMP3 cortisol CD40 peptide YY stem cell factor CD40 peptide YY TNF RII CD40 peptide YY AXL CD40 peptide YY Eotaxin 3 CD40 peptide YY FABP CD40 peptide YY FGF basic CD40 peptide YY myoglobin CD40 peptide YY resistin CD40 peptide YY TRAIL R3 CD40 peptide YY endothilin 1 CD40 peptide YY NrCAM CD40 peptide YY Tenascin C CD40 peptide YY VCAM1 CD40 peptide YY cortisol CD40 stem cell factor TNF RII CD40 stem cell factor AXL CD40 stem cell factor Eotaxin 3 CD40 stem cell factor FABP CD40 stem cell factor FGF basic CD40 stem cell factor myoglobin CD40 stem cell factor resistin CD40 stem cell factor TRAIL R3 CD40 stem cell factor endothilin 1 CD40 stem cell factor NrCAM CD40 stem cell factor Tenascin C CD40 stem cell factor VCAM1 CD40 stem cell factor cortisol CD40 TNF RII AXL CD40 TNF RII Eotaxin 3 CD40 TNF RII FABP CD40 TNF RII FGF basic CD40 TNF RII myoglobin CD40 TNF RII resistin CD40 TNF RII TRAIL R3 CD40 TNF RII endothilin 1 CD40 TNF RII NrCAM CD40 TNF RII Tenascin C CD40 TNF RII VCAM1 CD40 TNF RII cortisol CD40 AXL Eotaxin 3 CD40 AXL FABP CD40 AXL FGF basic CD40 AXL myoglobin CD40 AXL resistin CD40 AXL TRAIL R3 CD40 AXL endothilin 1 CD40 AXL NrCAM CD40 AXL Tenascin C CD40 AXL VCAM1 CD40 AXL cortisol CD40 Eotaxin 3 FABP CD40 Eotaxin 3 FGF basic CD40 Eotaxin 3 myoglobin CD40 Eotaxin 3 resistin CD40 Eotaxin 3 TRAIL R3 CD40 Eotaxin 3 endothilin 1 CD40 Eotaxin 3 NrCAM CD40 Eotaxin 3 Tenascin C CD40 Eotaxin 3 VCAM1 CD40 Eotaxin 3 cortisol CD40 FABP FGF basic CD40 FABP myoglobin CD40 FABP resistin CD40 FABP TRAIL R3 CD40 FABP endothilin 1 CD40 FABP NrCAM CD40 FABP Tenascin C CD40 FABP VCAM1 CD40 FABP cortisol CD40 FGF basic myoglobin CD40 FGF basic resistin CD40 FGF basic TRAIL R3 CD40 FGF basic endothilin 1 CD40 FGF basic NrCAM CD40 FGF basic Tenascin C CD40 FGF basic VCAM1 CD40 FGF basic cortisol CD40 myoglobin resistin CD40 myoglobin TRAIL R3 CD40 myoglobin endothilin 1 CD40 myoglobin NrCAM CD40 myoglobin Tenascin C CD40 myoglobin VCAM1 CD40 myoglobin cortisol CD40 resistin TRAIL R3 CD40 resistin endothilin 1 CD40 resistin NrCAM CD40 resistin Tenascin C CD40 resistin VCAM1 CD40 resistin cortisol CD40 TRAIL R3 endothilin 1 CD40 TRAIL R3 NrCAM CD40 TRAIL R3 Tenascin C CD40 TRAIL R3 VCAM1 CD40 TRAIL R3 cortisol CD40 endothilin 1 NrCAM CD40 endothilin 1 Tenascin C CD40 endothilin 1 VCAM1 CD40 endothilin 1 cortisol CD40 NrCAM Tenascin C CD40 NrCAM VCAM1 CD40 NrCAM cortisol CD40 Tenascin C VCAM1 CD40 Tenascin C cortisol CD40 VCAM1 cortisol IGF BP2 MMP3 peptide YY IGF BP2 MMP3 stem cell factor IGF BP2 MMP3 TNF RII IGF BP2 MMP3 AXL IGF BP2 MMP3 Eotaxin 3 IGF BP2 MMP3 FABP IGF BP2 MMP3 FGF basic IGF BP2 MMP3 myoglobin IGF BP2 MMP3 resistin IGF BP2 MMP3 TRAIL R3 IGF BP2 MMP3 endothilin 1 IGF BP2 MMP3 NrCAM IGF BP2 MMP3 Tenascin C IGF BP2 MMP3 VCAM1 IGF BP2 MMP3 cortisol IGF BP2 peptide YY stem cell factor IGF BP2 peptide YY TNF RII IGF BP2 peptide YY AXL IGF BP2 peptide YY Eotaxin 3 IGF BP2 peptide YY FABP IGF BP2 peptide YY FGF basic IGF BP2 peptide YY myoglobin IGF BP2 peptide YY resistin IGF BP2 peptide YY TRAIL R3 IGF BP2 peptide YY endothilin 1 IGF BP2 peptide YY NrCAM IGF BP2 peptide YY Tenascin C IGF BP2 peptide YY VCAM1 IGF BP2 peptide YY cortisol IGF BP2 stem cell factor TNF RII IGF BP2 stem cell factor AXL IGF BP2 stem cell factor Eotaxin 3 IGF BP2 stem cell factor FABP IGF BP2 stem cell factor FGF basic IGF BP2 stem cell factor myoglobin IGF BP2 stem cell factor resistin IGF BP2 stem cell factor TRAIL R3 IGF BP2 stem cell factor endothilin 1 IGF BP2 stem cell factor NrCAM IGF BP2 stem cell factor Tenascin C IGF BP2 stem cell factor VCAM1 IGF BP2 stem cell factor cortisol IGF BP2 TNF RII AXL IGF BP2 TNF RII Eotaxin 3 IGF BP2 TNF RII FABP IGF BP2 TNF RII FGF basic IGF BP2 TNF RII myoglobin IGF BP2 TNF RII resistin IGF BP2 TNF RII TRAIL R3 IGF BP2 TNF RII endothilin 1 IGF BP2 TNF RII NrCAM IGF BP2 TNF RII Tenascin C IGF BP2 TNF RII VCAM1 IGF BP2 TNF RII cortisol IGF BP2 AXL Eotaxin 3 IGF BP2 AXL FABP IGF BP2 AXL FGF basic IGF BP2 AXL myoglobin IGF BP2 AXL resistin IGF BP2 AXL TRAIL R3 IGF BP2 AXL endothilin 1 IGF BP2 AXL NrCAM IGF BP2 AXL Tenascin C IGF BP2 AXL VCAM1 IGF BP2 AXL cortisol IGF BP2 Eotaxin 3 FABP IGF BP2 Eotaxin 3 FGF basic IGF BP2 Eotaxin 3 myoglobin IGF BP2 Eotaxin 3 resistin IGF BP2 Eotaxin 3 TRAIL R3 IGF BP2 Eotaxin 3 endothilin 1 IGF BP2 Eotaxin 3 NrCAM IGF BP2 Eotaxin 3 Tenascin C IGF BP2 Eotaxin 3 VCAM1 IGF BP2 Eotaxin 3 cortisol IGF BP2 FABP FGF basic IGF BP2 FABP myoglobin IGF BP2 FABP resistin IGF BP2 FABP TRAIL R3 IGF BP2 FABP endothilin 1 IGF BP2 FABP NrCAM IGF BP2 FABP Tenascin C IGF BP2 FABP VCAM1 IGF BP2 FABP cortisol IGF BP2 FGF basic myoglobin IGF BP2 FGF basic resistin IGF BP2 FGF basic TRAIL R3 IGF BP2 FGF basic endothilin 1 IGF BP2 FGF basic NrCAM IGF BP2 FGF basic Tenascin C IGF BP2 FGF basic VCAM1 IGF BP2 FGF basic cortisol IGF BP2 myoglobin resistin IGF BP2 myoglobin TRAIL R3 IGF BP2 myoglobin endothilin 1 IGF BP2 myoglobin NrCAM IGF BP2 myoglobin Tenascin C IGF BP2 myoglobin VCAM1 IGF BP2 myoglobin cortisol IGF BP2 resistin TRAIL R3 IGF BP2 resistin endothilin 1 IGF BP2 resistin NrCAM IGF BP2 resistin Tenascin C IGF BP2 resistin VCAM1 IGF BP2 resistin cortisol IGF BP2 TRAIL R3 endothilin 1 IGF BP2 TRAIL R3 NrCAM IGF BP2 TRAIL R3 Tenascin C IGF BP2 TRAIL R3 VCAM1 IGF BP2 TRAIL R3 cortisol IGF BP2 endothilin 1 NrCAM IGF BP2 endothilin 1 Tenascin C IGF BP2 endothilin 1 VCAM1 IGF BP2 endothilin 1 cortisol IGF BP2 NrCAM Tenascin C IGF BP2 NrCAM VCAM1 IGF BP2 NrCAM cortisol IGF BP2 Tenascin C VCAM1 IGF BP2 Tenascin C cortisol IGF BP2 VCAM1 cortisol MMP3 peptide YY stem cell factor MMP3 peptide YY TNF RII MMP3 peptide YY AXL MMP3 peptide YY Eotaxin 3 MMP3 peptide YY FABP MMP3 peptide YY FGF basic MMP3 peptide YY myoglobin MMP3 peptide YY resistin MMP3 peptide YY TRAIL R3 MMP3 peptide YY endothilin 1 MMP3 peptide YY NrCAM MMP3 peptide YY Tenascin C MMP3 peptide YY VCAM1 MMP3 peptide YY cortisol MMP3 stem cell factor TNF RII MMP3 stem cell factor AXL MMP3 stem cell factor Eotaxin 3 MMP3 stem cell factor FABP MMP3 stem cell factor FGF basic MMP3 stem cell factor myoglobin MMP3 stem cell factor resistin MMP3 stem cell factor TRAIL R3 MMP3 stem cell factor endothilin 1 MMP3 stem cell factor NrCAM MMP3 stem cell factor Tenascin C MMP3 stem cell factor VCAM1 MMP3 stem cell factor cortisol MMP3 TNF RII AXL MMP3 TNF RII Eotaxin 3 MMP3 TNF RII FABP MMP3 TNF RII FGF basic MMP3 TNF RII myoglobin MMP3 TNF RII resistin MMP3 TNF RII TRAIL R3 MMP3 TNF RII endothilin 1 MMP3 TNF RII NrCAM MMP3 TNF RII Tenascin C MMP3 TNF RII VCAM1 MMP3 TNF RII cortisol MMP3 AXL Eotaxin 3 MMP3 AXL FABP MMP3 AXL FGF basic MMP3 AXL myoglobin MMP3 AXL resistin MMP3 AXL TRAIL R3 MMP3 AXL endothilin 1 MMP3 AXL NrCAM MMP3 AXL Tenascin C MMP3 AXL VCAM1 MMP3 AXL cortisol MMP3 Eotaxin 3 FABP MMP3 Eotaxin 3 FGF basic MMP3 Eotaxin 3 myoglobin MMP3 Eotaxin 3 resistin MMP3 Eotaxin 3 TRAIL R3 MMP3 Eotaxin 3 endothilin 1 MMP3 Eotaxin 3 NrCAM MMP3 Eotaxin 3 Tenascin C MMP3 Eotaxin 3 VCAM1 MMP3 Eotaxin 3 cortisol MMP3 FABP FGF basic MMP3 FABP myoglobin MMP3 FABP resistin MMP3 FABP TRAIL R3 MMP3 FABP endothilin 1 MMP3 FABP NrCAM MMP3 FABP Tenascin C MMP3 FABP VCAM1 MMP3 FABP cortisol MMP3 FGF basic myoglobin MMP3 FGF basic resistin MMP3 FGF basic TRAIL R3 MMP3 FGF basic endothilin 1 MMP3 FGF basic NrCAM MMP3 FGF basic Tenascin C MMP3 FGF basic VCAM1 MMP3 FGF basic cortisol MMP3 myoglobin resistin MMP3 myoglobin TRAIL R3 MMP3 myoglobin endothilin 1 MMP3 myoglobin NrCAM MMP3 myoglobin Tenascin C MMP3 myoglobin VCAM1 MMP3 myoglobin cortisol MMP3 resistin TRAIL R3 MMP3 resistin endothilin 1 MMP3 resistin NrCAM MMP3 resistin Tenascin C MMP3 resistin VCAM1 MMP3 resistin cortisol MMP3 TRAIL R3 endothilin 1 MMP3 TRAIL R3 NrCAM MMP3 TRAIL R3 Tenascin C MMP3 TRAIL R3 VCAM1 MMP3 TRAIL R3 cortisol MMP3 endothilin 1 NrCAM MMP3 endothilin 1 Tenascin C MMP3 endothilin 1 VCAM1 MMP3 endothilin 1 cortisol MMP3 NrCAM Tenascin C MMP3 NrCAM VCAM1 MMP3 NrCAM cortisol MMP3 Tenascin C VCAM1 MMP3 Tenascin C cortisol MMP3 VCAM1 cortisol peptide YY stem cell factor TNF RII peptide YY stem cell factor AXL peptide YY stem cell factor Eotaxin 3 peptide YY stem cell factor FABP peptide YY stem cell factor FGF basic peptide YY stem cell factor myoglobin peptide YY stem cell factor resistin peptide YY stem cell factor TRAIL R3 peptide YY stem cell factor endothilin 1 peptide YY stem cell factor NrCAM peptide YY stem cell factor Tenascin C peptide YY stem cell factor VCAM1 peptide YY stem cell factor cortisol peptide YY TNF RII AXL peptide YY TNF RII Eotaxin 3 peptide YY TNF RII FABP peptide YY TNF RII FGF basic peptide YY TNF RII myoglobin peptide YY TNF RII resistin peptide YY TNF RII TRAIL R3 peptide YY TNF RII endothilin 1 peptide YY TNF RII NrCAM peptide YY TNF RII Tenascin C peptide YY TNF RII VCAM1 peptide YY TNF RII cortisol peptide YY AXL Eotaxin 3 peptide YY AXL FABP peptide YY AXL FGF basic peptide YY AXL myoglobin peptide YY AXL resistin peptide YY AXL TRAIL R3 peptide YY AXL endothilin 1 peptide YY AXL NrCAM peptide YY AXL Tenascin C peptide YY AXL VCAM1 peptide YY AXL cortisol peptide YY Eotaxin 3 FABP peptide YY Eotaxin 3 FGF basic peptide YY Eotaxin 3 myoglobin peptide YY Eotaxin 3 resistin peptide YY Eotaxin 3 TRAIL R3 peptide YY Eotaxin 3 endothilin 1 peptide YY Eotaxin 3 NrCAM peptide YY Eotaxin 3 Tenascin C peptide YY Eotaxin 3 VCAM1 peptide YY Eotaxin 3 cortisol peptide YY FABP FGF basic peptide YY FABP myoglobin peptide YY FABP resistin peptide YY FABP TRAIL R3 peptide YY FABP endothilin 1 peptide YY FABP NrCAM peptide YY FABP Tenascin C peptide YY FABP VCAM1 peptide YY FABP cortisol peptide YY FGF basic myoglobin peptide YY FGF basic resistin peptide YY FGF basic TRAIL R3 peptide YY FGF basic endothilin 1 peptide YY FGF basic NrCAM peptide YY FGF basic Tenascin C peptide YY FGF basic VCAM1 peptide YY FGF basic cortisol peptide YY myoglobin resistin peptide YY myoglobin TRAIL R3 peptide YY myoglobin endothilin 1 peptide YY myoglobin NrCAM peptide YY myoglobin Tenascin C peptide YY myoglobin VCAM1 peptide YY myoglobin cortisol peptide YY resistin TRAIL R3 peptide YY resistin endothilin 1 peptide YY resistin NrCAM peptide YY resistin Tenascin C peptide YY resistin VCAM1 peptide YY resistin cortisol peptide YY TRAIL R3 endothilin 1 peptide YY TRAIL R3 NrCAM peptide YY TRAIL R3 Tenascin C peptide YY TRAIL R3 VCAM1 peptide YY TRAIL R3 cortisol peptide YY endothilin 1 NrCAM peptide YY endothilin 1 Tenascin C peptide YY endothilin 1 VCAM1 peptide YY endothilin 1 cortisol peptide YY NrCAM Tenascin C peptide YY NrCAM VCAM1 peptide YY NrCAM cortisol peptide YY Tenascin C VCAM1 peptide YY Tenascin C cortisol peptide YY VCAM1 cortisol stem cell factor TNF RII AXL stem cell factor TNF RII Eotaxin 3 stem cell factor TNF RII FABP stem cell factor TNF RII FGF basic stem cell factor TNF RII myoglobin stem cell factor TNF RII resistin stem cell factor TNF RII TRAIL R3 stem cell factor TNF RII endothilin 1 stem cell factor TNF RII NrCAM stem cell factor TNF RII Tenascin C stem cell factor TNF RII VCAM1 stem cell factor TNF RII cortisol stem cell factor AXL Eotaxin 3 stem cell factor AXL FABP stem cell factor AXL FGF basic stem cell factor AXL myoglobin stem cell factor AXL resistin stem cell factor AXL TRAIL R3 stem cell factor AXL endothilin 1 stem cell factor AXL NrCAM stem cell factor AXL Tenascin C stem cell factor AXL VCAM1 stem cell factor AXL cortisol stem cell factor Eotaxin 3 FABP stem cell factor Eotaxin 3 FGF basic stem cell factor Eotaxin 3 myoglobin stem cell factor Eotaxin 3 resistin stem cell factor Eotaxin 3 TRAIL R3 stem cell factor Eotaxin 3 endothilin 1 stem cell factor Eotaxin 3 NrCAM stem cell factor Eotaxin 3 Tenascin C stem cell factor Eotaxin 3 VCAM1 stem cell factor Eotaxin 3 cortisol stem cell factor FABP FGF basic stem cell factor FABP myoglobin stem cell factor FABP resistin stem cell factor FABP TRAIL R3 stem cell factor FABP endothilin 1 stem cell factor FABP NrCAM stem cell factor FABP Tenascin C stem cell factor FABP VCAM1 stem cell factor FABP cortisol stem cell factor FGF basic myoglobin stem cell factor FGF basic resistin stem cell factor FGF basic TRAIL R3 stem cell factor FGF basic endothilin 1 stem cell factor FGF basic NrCAM stem cell factor FGF basic Tenascin C stem cell factor FGF basic VCAM1 stem cell factor FGF basic cortisol stem cell factor myoglobin resistin stem cell factor myoglobin TRAIL R3 stem cell factor myoglobin endothilin 1 stem cell factor myoglobin NrCAM stem cell factor myoglobin Tenascin C stem cell factor myoglobin VCAM1 stem cell factor myoglobin cortisol stem cell factor resistin TRAIL R3 stem cell factor resistin endothilin 1 stem cell factor resistin NrCAM stem cell factor resistin Tenascin C stem cell factor resistin VCAM1 stem cell factor resistin cortisol stem cell factor TRAIL R3 endothilin 1 stem cell factor TRAIL R3 NrCAM stem cell factor TRAIL R3 Tenascin C stem cell factor TRAIL R3 VCAM1 stem cell factor TRAIL R3 cortisol stem cell factor endothilin 1 NrCAM stem cell factor endothilin 1 Tenascin C stem cell factor endothilin 1 VCAM1 stem cell factor endothilin 1 cortisol stem cell factor NrCAM Tenascin C stem cell factor NrCAM VCAM1 stem cell factor NrCAM cortisol stem cell factor Tenascin C VCAM1 stem cell factor Tenascin C cortisol stem cell factor VCAM1 cortisol TNF RII AXL Eotaxin 3 TNF RII AXL FABP TNF RII AXL FGF basic TNF RII AXL myoglobin TNF RII AXL resistin TNF RII AXL TRAIL R3 TNF RII AXL endothilin 1 TNF RII AXL NrCAM TNF RII AXL Tenascin C TNF RII AXL VCAM1 TNF RII AXL cortisol TNF RII Eotaxin 3 FABP TNF RII Eotaxin 3 FGF basic TNF RII Eotaxin 3 myoglobin TNF RII Eotaxin 3 resistin TNF RII Eotaxin 3 TRAIL R3 TNF RII Eotaxin 3 endothilin 1 TNF RII Eotaxin 3 NrCAM TNF RII Eotaxin 3 Tenascin C TNF RII Eotaxin 3 VCAM1 TNF RII Eotaxin 3 cortisol TNF RII FABP FGF basic TNF RII FABP myoglobin TNF RII FABP resistin TNF RII FABP TRAIL R3 TNF RII FABP endothilin 1 TNF RII FABP NrCAM TNF RII FABP Tenascin C TNF RII FABP VCAM1 TNF RII FABP cortisol TNF RII FGF basic myoglobin TNF RII FGF basic resistin TNF RII FGF basic TRAIL R3 TNF RII FGF basic endothilin 1 TNF RII FGF basic NrCAM TNF RII FGF basic Tenascin C TNF RII FGF basic VCAM1 TNF RII FGF basic cortisol TNF RII myoglobin resistin TNF RII myoglobin TRAIL R3 TNF RII myoglobin endothilin 1 TNF RII myoglobin NrCAM TNF RII myoglobin Tenascin C TNF RII myoglobin VCAM1 TNF RII myoglobin cortisol TNF RII resistin TRAIL R3 TNF RII resistin endothilin 1 TNF RII resistin NrCAM TNF RII resistin Tenascin C TNF RII resistin VCAM1 TNF RII resistin cortisol TNF RII TRAIL R3 endothilin 1 TNF RII TRAIL R3 NrCAM TNF RII TRAIL R3 Tenascin C TNF RII TRAIL R3 VCAM1 TNF RII TRAIL R3 cortisol TNF RII endothilin 1 NrCAM TNF RII endothilin 1 Tenascin C TNF RII endothilin 1 VCAM1 TNF RII endothilin 1 cortisol TNF RII NrCAM Tenascin C TNF RII NrCAM VCAM1 TNF RII NrCAM cortisol TNF RII Tenascin C VCAM1 TNF RII Tenascin C cortisol TNF RII VCAM1 cortisol AXL Eotaxin 3 FABP AXL Eotaxin 3 FGF basic AXL Eotaxin 3 myoglobin AXL Eotaxin 3 resistin AXL Eotaxin 3 TRAIL R3 AXL Eotaxin 3 endothilin 1 AXL Eotaxin 3 NrCAM AXL Eotaxin 3 Tenascin C AXL Eotaxin 3 VCAM1 AXL Eotaxin 3 cortisol AXL FABP FGF basic AXL FABP myoglobin AXL FABP resistin AXL FABP TRAIL R3 AXL FABP endothilin 1 AXL FABP NrCAM AXL FABP Tenascin C AXL FABP VCAM1 AXL FABP cortisol AXL FGF basic myoglobin AXL FGF basic resistin AXL FGF basic TRAIL R3 AXL FGF basic endothilin 1 AXL FGF basic NrCAM AXL FGF basic Tenascin C AXL FGF basic VCAM1 AXL FGF basic cortisol AXL myoglobin resistin AXL myoglobin TRAIL R3 AXL myoglobin endothilin 1 AXL myoglobin NrCAM AXL myoglobin Tenascin C AXL myoglobin VCAM1 AXL myoglobin cortisol AXL resistin TRAIL R3 AXL resistin endothilin 1 AXL resistin NrCAM AXL resistin Tenascin C AXL resistin VCAM1 AXL resistin cortisol AXL TRAIL R3 endothilin 1 AXL TRAIL R3 NrCAM AXL TRAIL R3 Tenascin C AXL TRAIL R3 VCAM1 AXL TRAIL R3 cortisol AXL endothilin 1 NrCAM AXL endothilin 1 Tenascin C AXL endothilin 1 VCAM1 AXL endothilin 1 cortisol AXL NrCAM Tenascin C AXL NrCAM VCAM1 AXL NrCAM cortisol AXL Tenascin C VCAM1 AXL Tenascin C cortisol AXL VCAM1 cortisol Eotaxin 3 FABP FGF basic Eotaxin 3 FABP myoglobin Eotaxin 3 FABP resistin Eotaxin 3 FABP TRAIL R3 Eotaxin 3 FABP endothilin 1 Eotaxin 3 FABP NrCAM Eotaxin 3 FABP Tenascin C Eotaxin 3 FABP VCAM1 Eotaxin 3 FABP cortisol Eotaxin 3 FGF basic myoglobin Eotaxin 3 FGF basic resistin Eotaxin 3 FGF basic TRAIL R3 Eotaxin 3 FGF basic endothilin 1 Eotaxin 3 FGF basic NrCAM Eotaxin 3 FGF basic Tenascin C Eotaxin 3 FGF basic VCAM1 Eotaxin 3 FGF basic cortisol Eotaxin 3 myoglobin resistin Eotaxin 3 myoglobin TRAIL R3 Eotaxin 3 myoglobin endothilin 1 Eotaxin 3 myoglobin NrCAM Eotaxin 3 myoglobin Tenascin C Eotaxin 3 myoglobin VCAM1 Eotaxin 3 myoglobin cortisol Eotaxin 3 resistin TRAIL R3 Eotaxin 3 resistin endothilin 1 Eotaxin 3 resistin NrCAM Eotaxin 3 resistin Tenascin C Eotaxin 3 resistin VCAM1 Eotaxin 3 resistin cortisol Eotaxin 3 TRAIL R3 endothilin 1 Eotaxin 3 TRAIL R3 NrCAM Eotaxin 3 TRAIL R3 Tenascin C Eotaxin 3 TRAIL R3 VCAM1 Eotaxin 3 TRAIL R3 cortisol Eotaxin 3 endothilin 1 NrCAM Eotaxin 3 endothilin 1 Tenascin C Eotaxin 3 endothilin 1 VCAM1 Eotaxin 3 endothilin 1 cortisol Eotaxin 3 NrCAM Tenascin C Eotaxin 3 NrCAM VCAM1 Eotaxin 3 NrCAM cortisol Eotaxin 3 Tenascin C VCAM1 Eotaxin 3 Tenascin C cortisol Eotaxin 3 VCAM1 cortisol FABP FGF basic myoglobin FABP FGF basic resistin FABP FGF basic TRAIL R3 FABP FGF basic endothilin 1 FABP FGF basic NrCAM FABP FGF basic Tenascin C FABP FGF basic VCAM1 FABP FGF basic cortisol FABP myoglobin resistin FABP myoglobin TRAIL R3 FABP myoglobin endothilin 1 FABP myoglobin NrCAM FABP myoglobin Tenascin C FABP myoglobin VCAM1 FABP myoglobin cortisol FABP resistin TRAIL R3 FABP resistin endothilin 1 FABP resistin NrCAM FABP resistin Tenascin C FABP resistin VCAM1 FABP resistin cortisol FABP TRAIL R3 endothilin 1 FABP TRAIL R3 NrCAM FABP TRAIL R3 Tenascin C FABP TRAIL R3 VCAM1 FABP TRAIL R3 cortisol FABP endothilin 1 NrCAM FABP endothilin 1 Tenascin C FABP endothilin 1 VCAM1 FABP endothilin 1 cortisol FABP NrCAM Tenascin C FABP NrCAM VCAM1 FABP NrCAM cortisol FABP Tenascin C VCAM1 FABP Tenascin C cortisol FABP VCAM1 cortisol FGF basic myoglobin resistin FGF basic myoglobin TRAIL R3 FGF basic myoglobin endothilin 1 FGF basic myoglobin NrCAM FGF basic myoglobin Tenascin C FGF basic myoglobin VCAM1 FGF basic myoglobin cortisol FGF basic resistin TRAIL R3 FGF basic resistin endothilin 1 FGF basic resistin NrCAM FGF basic resistin Tenascin C FGF basic resistin VCAM1 FGF basic resistin cortisol FGF basic TRAIL R3 endothilin 1 FGF basic TRAIL R3 NrCAM FGF basic TRAIL R3 Tenascin C FGF basic TRAIL R3 VCAM1 FGF basic TRAIL R3 cortisol FGF basic endothilin 1 NrCAM FGF basic endothilin 1 Tenascin C FGF basic endothilin 1 VCAM1 FGF basic endothilin 1 cortisol FGF basic NrCAM Tenascin C FGF basic NrCAM VCAM1 FGF basic NrCAM cortisol FGF basic Tenascin C VCAM1 FGF basic Tenascin C cortisol FGF basic VCAM1 cortisol myoglobin resistin TRAIL R3 myoglobin resistin endothilin 1 myoglobin resistin NrCAM myoglobin resistin Tenascin C myoglobin resistin VCAM1 myoglobin resistin cortisol myoglobin TRAIL R3 endothilin 1 myoglobin TRAIL R3 NrCAM myoglobin TRAIL R3 Tenascin C myoglobin TRAIL R3 VCAM1 myoglobin TRAIL R3 cortisol myoglobin endothilin 1 NrCAM myoglobin endothilin 1 Tenascin C myoglobin endothilin 1 VCAM1 myoglobin endothilin 1 cortisol myoglobin NrCAM Tenascin C myoglobin NrCAM VCAM1 myoglobin NrCAM cortisol myoglobin Tenascin C VCAM1 myoglobin Tenascin C cortisol myoglobin VCAM1 cortisol resistin TRAIL R3 endothilin 1 resistin TRAIL R3 NrCAM resistin TRAIL R3 Tenascin C resistin TRAIL R3 VCAM1 resistin TRAIL R3 cortisol resistin endothilin 1 NrCAM resistin endothilin 1 Tenascin C resistin endothilin 1 VCAM1 resistin endothilin 1 cortisol resistin NrCAM Tenascin C resistin NrCAM VCAM1 resistin NrCAM cortisol resistin Tenascin C VCAM1 resistin Tenascin C cortisol resistin VCAM1 cortisol TRAIL R3 endothilin 1 NrCAM TRAIL R3 endothilin 1 Tenascin C TRAIL R3 endothilin 1 VCAM1 TRAIL R3 endothilin 1 cortisol TRAIL R3 NrCAM Tenascin C TRAIL R3 NrCAM VCAM1 TRAIL R3 NrCAM cortisol TRAIL R3 Tenascin C VCAM1 TRAIL R3 Tenascin C cortisol TRAIL R3 VCAM1 cortisol endothilin 1 NrCAM Tenascin C endothilin 1 NrCAM VCAM1 endothilin 1 NrCAM cortisol endothilin 1 Tenascin C VCAM1 endothilin 1 Tenascin C cortisol endothilin 1 VCAM1 cortisol NrCAM Tenascin C VCAM1 NrCAM Tenascin C cortisol NrCAM VCAM1 cortisol Tenascin C VCAM1 cortisol

III. Test Sample

The method for diagnosing, monitoring, or determining a renal disorder involves determining the presence of sample analytes in a test sample. A test sample, as defined herein, is an amount of bodily fluid taken from a mammal. Non-limiting examples of bodily fluids include urine, blood, plasma, serum, saliva, semen, perspiration, tears, mucus, and tissue lysates. In an exemplary embodiment, the bodily fluid contained in the test sample is urine, plasma, or serum.

(a) Mammals

A mammal, as defined herein, is any organism that is a member of the class Mammalia. Non-limiting examples of mammals appropriate for the various embodiments may include humans, apes, monkeys, rats, mice, dogs, cats, pigs, and livestock including cattle and oxen. In an exemplary embodiment, the mammal is a human.

(b) Devices and Methods of Taking Bodily Fluids from Mammals

The bodily fluids of the test sample may be taken from the mammal using any known device or method so long as the analytes to be measured by the multiplexed assay are not rendered undetectable by the multiplexed assay. Non-limiting examples of devices or methods suitable for taking bodily fluid from a mammal include urine sample cups, urethral catheters, swabs, hypodermic needles, thin needle biopsies, hollow needle biopsies, punch biopsies, metabolic cages, and aspiration.

In order to adjust the expected concentrations of the sample analytes in the test sample to fall within the dynamic range of the multiplexed assay, the test sample may be diluted to reduce the concentration of the sample analytes prior to analysis. The degree of dilution may depend on a variety of factors including but not limited to the type of multiplexed assay used to measure the analytes, the reagents utilized in the multiplexed assay, and the type of bodily fluid contained in the test sample. In one embodiment, the test sample is diluted by adding a volume of diluent ranging from about ½ of the original test sample volume to about 50,000 times the original test sample volume.

In one exemplary embodiment, if the test sample is human urine and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 100 times the original test sample volume prior to analysis. In another exemplary embodiment, if the test sample is human serum and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 5 times the original test sample volume prior to analysis. In yet another exemplary embodiment, if the test sample is human plasma and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 2,000 times the original test sample volume prior to analysis.

The diluent may be any fluid that does not interfere with the function of the multiplexed assay used to measure the concentration of the analytes in the test sample. Non-limiting examples of suitable diluents include deionized water, distilled water, saline solution, Ringer's solution, phosphate buffered saline solution, TRIS-buffered saline solution, standard saline citrate, and HEPES-buffered saline.

IV. Multiplexed Assay Device

In one embodiment, the concentration of a combination of sample analytes is measured using a multiplexed assay device capable of measuring up to 189 of the biomarker analytes. A multiplexed assay device, as defined herein, is an assay capable of simultaneously determining the concentration of three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more of the biomarker analytes using a single device and/or method. Any known method of measuring the concentration of the biomarker analytes may be used for the multiplexed assay device. Non-limiting examples of measurement methods suitable for the multiplexed assay device include electrophoresis, mass spectrometry, protein microarrays, surface plasmon resonance, and immunoassays including, but not limited to western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA) methods, vibrational detection using MicroElectroMagnetic Devices (MEMS), and particle-based capture-sandwich immunoassays.

(a) Multiplexed Immunoassay Device

In one embodiment, the concentrations of the analytes in the test sample are measured using a multiplexed immunoassay device that utilizes capture antibodies marked with indicators to determine the concentration of the sample analytes.

(i) Capture Antibodies

In the same embodiment, the multiplexed immunoassay device includes three or more capture antibodies. Capture antibodies, as defined herein, are antibodies in which the antigenic determinant is one of the Biomarker Analytes known in the art to have a documented association with early renal damage in humans. The biomarker analytes include, but are note limited to alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF. Each of the at least three capture antibodies has a unique antigenic determinant that is one of the biomarker analytes. When contacted with the test sample, the capture antibodies form antigen-antibody complexes in which the analytes serve as antigens.

The term “antibody,” as used herein, encompasses a monoclonal ab, an antibody fragment, a chimeric antibody, and a single-chain antibody.

In some embodiments, the capture antibodies may be attached to a platform or other substrate having a contact surface in order to immobilize any analytes captured by the capture antibodies. The platform generally incorporates a porous material for immobilizing the analytes. Non-limiting examples of suitable substrates include paper, nitrocellulose, cellulose, glass, glass fiber mesh, silica gel, synthetic resins, or plastic strips, beads, or surfaces, such as the inner surface of the well of a microtitration tray. Suitable beads may include polystyrene or latex microspheres.

(ii) indicators

In one embodiment of the multiplexed immunoassay device, an indicator is attached to each of the three or more capture antibodies. The indicator, as defined herein, is any compound that registers a measurable change to indicate the presence of one of the sample analytes when bound to one of the capture antibodies. Non-limiting examples of indicators include visual indicators and electrochemical indicators.

Visual indicators, as defined herein, are compounds that register a change by reflecting a limited subset of the wavelengths of light illuminating the indicator, by fluorescing light after being illuminated, or by emitting light via chemiluminescence. The change registered by visual indicators may be in the visible light spectrum, in the infrared spectrum, or in the ultraviolet spectrum. Non-limiting examples of visual indicators suitable for the multiplexed immunoassay device include nanoparticulate gold, organic particles such as polyurethane or latex microspheres loaded with dye compounds, carbon black, fluorophores, phycoerythrin, radioactive isotopes, nanoparticles, quantum dots, and enzymes such as horseradish peroxidase or alkaline phosphatase that react with a chemical substrate to form a colored or chemiluminescent product.

Electrochemical indicators, as defined herein, are compounds that register a change by altering an electrical property. The changes registered by electrochemical indicators may be an alteration in conductivity, resistance, capacitance, current conducted in response to an applied voltage, or voltage required to achieve a desired current. Non-limiting examples of electrochemical indicators include redox species such as ascorbate (vitamin C), vitamin E, glutathione, polyphenols, catechols, quercetin, phytoestrogens, penicillin, carbazole, murranes, phenols, carbonyls, benzoates, and trace metal ions such as nickel, copper, cadmium, iron and mercury.

In this same embodiment, the test sample containing a combination of three or more sample analytes is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as the antigens. After removing any uncomplexed capture antibodies, the concentrations of the three or more analytes are determined by measuring the change registered by the indicators attached to the capture antibodies.

In one exemplary embodiment, the indicators are polyurethane or latex microspheres loaded with dye compounds and phycoerythrin.

(b) Multiplexed Sandwich Immunoassay Device

In another embodiment, the multiplexed immunoassay device has a sandwich assay format. In this embodiment, the multiplexed sandwich immunoassay device includes three or more capture antibodies as previously described. However, in this embodiment, each of the capture antibodies is attached to a capture agent that includes an antigenic moiety. The antigenic moiety serves as the antigenic determinant of a detection antibody, also included in the multiplexed immunoassay device of this embodiment. In addition, an indicator is attached to the detection antibody.

In this same embodiment, the test sample is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as antigens. The detection antibodies are then contacted with the test sample and allowed to form antigen-antibody complexes in which the capture agent serves as the antigen for the detection antibody. After removing any uncomplexed detection antibodies the concentration of the analytes are determined by measuring the changes registered by the indicators attached to the detection antibodies.

(c) Multiplexing Approaches

In the various embodiments of the multiplexed immunoassay devices, the concentrations of each of the sample analytes may be determined using any approach known in the art. In one embodiment, a single indicator compound is attached to each of the three or more antibodies. In addition, each of the capture antibodies having one of the sample analytes as an antigenic determinant is physically separated into a distinct region so that the concentration of each of the sample analytes may be determined by measuring the changes registered by the indicators in each physically separate region corresponding to each of the sample analytes.

In another embodiment, each antibody having one of the sample analytes as an antigenic determinant is marked with a unique indicator. In this manner, a unique indicator is attached to each antibody having a single sample analyte as its antigenic determinant. In this embodiment, all antibodies may occupy the same physical space. The concentration of each sample analyte is determined by measuring the change registered by the unique indicator attached to the antibody having the sample analyte as an antigenic determinant.

(d) Microsphere-Based Capture-Sandwich Immunoassay Device

In an exemplary embodiment, the multiplexed immunoassay device is a microsphere-based capture-sandwich immunoassay device. In this embodiment, the device includes a mixture of three or more capture-antibody microspheres, in which each capture-antibody microsphere corresponds to one of the biomarker analytes. Each capture-antibody microsphere includes a plurality of capture antibodies attached to the outer surface of the microsphere. In this same embodiment, the antigenic determinant of all of the capture antibodies attached to one microsphere is the same biomarker analyte.

In this embodiment of the device, the microsphere is a small polystyrene or latex sphere that is loaded with an indicator that is a dye compound. The microsphere may be between about 3 μm and about 5 μm in diameter. Each capture-antibody microsphere corresponding to one of the biomarker analytes is loaded with the same indicator. In this manner, each capture-antibody microsphere corresponding to a biomarker analyte is uniquely color-coded.

In this same exemplary embodiment, the multiplexed immunoassay device further includes three or more biotinylated detection antibodies in which the antigenic determinant of each biotinylated detection antibody is one of the biomarker analytes. The device further includes a plurality of streptaviden proteins complexed with a reporter compound. A reporter compound, as defined herein, is an indicator selected to register a change that is distinguishable from the indicators used to mark the capture-antibody microspheres.

The concentrations of the sample analytes may be determined by contacting the test sample with a mixture of capture-antigen microspheres corresponding to each sample analyte to be measured. The sample analytes are allowed to form antigen-antibody complexes in which a sample analyte serves as an antigen and a capture antibody attached to the microsphere serves as an antibody. In this manner, the sample analytes are immobilized onto the capture-antigen microspheres. The biotinylated detection antibodies are then added to the test sample and allowed to form antigen-antibody complexes in which the analyte serves as the antigen and the biotinylated detection antibody serves as the antibody. The streptaviden-reporter complex is then added to the test sample and allowed to bind to the biotin moieties of the biotinylated detection antibodies. The antigen-capture microspheres may then be rinsed and filtered.

In this embodiment, the concentration of each analyte is determined by first measuring the change registered by the indicator compound embedded in the capture-antigen microsphere in order to identify the particular analyte. For each microsphere corresponding to one of the biomarker analytes, the quantity of analyte immobilized on the microsphere is determined by measuring the change registered by the reporter compound attached to the microsphere.

For example, the indicator embedded in the microspheres associated with one sample analyte may register an emission of orange light, and the reporter may register an emission of green light. In this example, a detector device may measure the intensity of orange light and green light separately. The measured intensity of the green light would determine the concentration of the analyte captured on the microsphere, and the intensity of the orange light would determine the specific analyte captured on the microsphere.

Any sensor device may be used to detect the changes registered by the indicators embedded in the microspheres and the changes registered by the reporter compound, so long as the sensor device is sufficiently sensitive to the changes registered by both indicator and reporter compound. Non-limiting examples of suitable sensor devices include spectrophotometers, photosensors, colorimeters, cyclic coulometry devices, and flow cytometers. In an exemplary embodiment, the sensor device is a flow cytometer.

(e) Vibrational Detection Device

In another exemplary embodiment, the multiplexed immunoassay device has a vibrational detection format using a MEMS. In this embodiment, the immunoassay device uses capture antibodies as previously described. However, in this embodiment, the capture antibodies are attached to a microscopic silicon microcantilever beam structure. The microcantilevers are micromechanical beams that are anchored at one end, such as diving spring boards that can be readily fabricated on silicon wafers and other materials. The microcantilever sensors are physical sensors that respond to surface stress changes due to chemical or biological processes. When fabricated with very small force constants, they can measure forces and stresses with extremely high sensitivity. The very small force constant of a cantilever allows detection not surface stress variation due to the binding of an analyte to the capture antibody on the microcantilever. Binding of the analyte results in a differential surface stress due to adsorption-induced forces, which manifests as a deflection which can be measured. The vibrational detection may be multiplexed. For more details, see Datar et al., MRS Bulletin (2009) 34:449-459 and Gaster et al., Nature Medicine (2009) 15:1327-1332, both of which are hereby incorporated by reference in their entireties.

It will be understood by one skilled in the art that the devices described herein, as well as all those embodiments within the scope of the current invention may be incorporated into a kit. Generally, the kit may include any of the devices described herein in addition to a collection apparatus suitable for collecting a sample of bodily fluid from the mammal. The collection apparatus may include, but it not limited to urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, aspiration needles, and combinations thereof.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1 Least Detectable Dose and Lower Limit of Quantitation of Assay for Analytes Associated with Renal Disorders

To assess the least detectable doses (LDD) and lower limits of quantitation (LLOQ) of a variety of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.

The concentrations of the analytes were measured using a capture-sandwich assay using antigen-specific antibodies. For each analyte, a range of standard sample dilutions ranging over about four orders of magnitude of analyte concentration were measured using the assay in order to obtain data used to construct a standard dose response curve. The dynamic range for each of the analytes, defined herein as the range of analyte concentrations measured to determine its dose response curve, is presented below.

To perform the assay, 5 μL of a diluted mixture of capture-antibody microspheres were mixed with 5 μL of blocker and 10 μL of pre-diluted standard sample in each of the wells of a hard-bottom microtiter plate. After incubating the hard-bottom plate for 1 hour, 10 μL of biotinylated detection antibody was added to each well, and then the hard-bottom plate was incubated for an additional hour. 10 μL of diluted streptavidin-phycoerythrin was added to each well and then the hard-bottom plate was incubated for another 60 minutes.

A filter-membrane microtiter plate was pre-wetted by adding 100 μL wash buffer, and then aspirated using a vacuum manifold device. The contents of the wells of the hard-bottom plate were then transferred to the corresponding wells of the filter-membrane plate. All wells of the hard-bottom plate were vacuum-aspirated and the contents were washed twice with 100 μL of wash buffer. After the second wash, 100 μL of wash buffer was added to each well, and then the washed microspheres were resuspended with thorough mixing. The plate was then analyzed using a Luminex 100 Analyzer (Luminex Corporation, Austin, Tex., USA). Dose response curves were constructed for each analyte by curve-fitting the median fluorescence intensity (MFI) measured from the assays of diluted standard samples containing a range of analyte concentrations.

The least detectable dose (LDD) was determined by adding three standard deviations to the average of the MFI signal measured for 20 replicate samples of blank standard solution (i.e. standard solution containing no analyte). The MFI signal was converted to an LDD concentration using the dose response curve and multiplied by a dilution factor of 2.

The lower limit of quantification (LLOQ), defined herein as the point at which the coefficient of variation (CV) for the analyte measured in the standard samples was 30%, was determined by the analysis of the measurements of increasingly diluted standard samples. For each analyte, the standard solution was diluted by 2 fold for 8 dilutions. At each stage of dilution, samples were assayed in triplicate, and the CV of the analyte concentration at each dilution was calculated and plotted as a function of analyte concentration. The LLOQ was interpolated from this plot and multiplied by a dilution factor of 2.

The LDD and LLOQ results for each analyte are summarized in Table 2:

TABLE 2 LDD, LLOQ, and Dynamic Range of Analyte Assay Dynamic Range Analyte Units LDD LLOQ minimum maximum Calbindin ng/mL 1.1 3.1 0.516 2580 Clusterin ng/mL 2.4 2.3 0.676 3378 CTGF ng/mL 1.3 3.8 0.0794 400 GST-alpha ng/mL 1.4 3.6 0.24 1,200 KIM-1 ng/mL 0.016 0.028 0.00478 24 VEGF pg/mL 4.4 20 8.76 44,000 β-2M μg/mL 0.012 0.018 0.0030 15 Cystatin C ng/mL 2.8 3.7 0.60 3,000 NGAL ng/mL 4.1 7.8 1.2 6,000 Osteopontin ng/mL 29 52 3.9 19,500 TIMP-1 ng/mL 0.71 1.1 0.073 365 A-1M μg/mL 0.059 0.29 0.042 210 THP μg/mL 0.46 0.30 0.16 800 TFF-3 μg/mL 0.06 0.097 0.060 300

The results of this experiment characterized the least detectible dose and the lower limit of quantification for fourteen analytes associated with various renal disorders using a capture-sandwich assay.

Example 2 Precision of Assay for Analytes Associated with Renal Disorders

To assess the precision of an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were measured in triplicate during three runs using the methods described in Example 1. The percent errors for each run at each concentration are presented in Table 3 for all of the analytes tested:

TABLE 3 Precision of Analyte Assay Average Run 2 Interrun concentration Run 1 Error Run 2 Error Analyte (ng/mL) Error (%) (%) Error (%) (%) Calbindin 4.0 6 2 6 13 36 5 3 2 7 281 1 6 0 3 Clusterin 4.4 4 9 2 6 39 5 1 6 8 229 1 3 0 2 CTGF 1.2 10 17 4 14 2.5 19 19 14 14 18 7 5 13 9 GST-alpha 3.9 14 7 5 10 16 13 7 10 11 42 1 16 6 8 KIM-1 0.035 2 0 5 13 0.32 4 5 2 8 2.9 0 5 7 4 VEGF 65 10 1 6 14 534 9 2 12 7 5,397 1 13 14 9 β-2M 0.040 6 1 8 5 0.43 2 2 0 10 6.7 6 5 11 6 Cystatin C 10.5 4 1 7 13 49 0 0 3 9 424 2 6 2 5 NGAL 18.1 11 3 6 13 147 0 0 6 5 1,070 5 1 2 5 Osteopontin 44 1 10 2 11 523 9 9 9 7 8,930 4 10 1 10 TIMP-1 2.2 13 6 3 13 26 1 1 4 14 130 1 3 1 4 A-1M 1.7 11 7 7 14 19 4 1 8 9 45 3 5 2 4 THP 9.4 3 10 11 11 15 3 7 8 6 37 4 5 0 5 TFF-3 0.3 13 3 11 12 4.2 5 8 5 7 1.2 3 7 0 13

The results of this experiment characterized the precision of a capture-sandwich assay for fourteen analytes associated with various renal disorders over a wide range of analyte concentrations. The precision of the assay varied between about 1% and about 15% error within a given run, and between about 5% and about 15% error between different runs. The percent errors summarized in Table 2 provide information concerning random error to be expected in an assay measurement caused by variations in technicians, measuring instruments, and times of measurement.

Example 3 Linearity of Assay for Analytes Associated with Renal Disorders

To assess the linearity of an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were measured in triplicate during three runs using the methods described in Example 1. Linearity of the assay used to measure each analyte was determined by measuring the concentrations of standard samples that were serially-diluted throughout the assay range. The % recovery was calculated as observed vs. expected concentration based on the dose-response curve. The results of the linearity analysis are summarized in Table 4.

TABLE 4 Linearity of Analyte Assay Expected Observed Recovery Analyte Dilution concentration concentration (%) Calbindin 1:2 61 61 100 (ng/mL) 1:4 30 32 106 1:8 15 17 110 Clusterin 1:2 41 41 100 (ng/mL) 1:4 21 24 116 1:8 10 11 111 CTGF 1:2 1.7 1.7 100 (ng/mL) 1:4 0.84 1.0 124 1:8 0.42 0.51 122 GST-alpha 1:2 25 25 100 (ng/mL) 1:4 12 14 115 1:8 6.2 8.0 129 KIM-1 1:2 0.87 0.87 100 (ng/mL) 1:4 0.41 0.41 101 1:8 0.21 0.19 93 VEGF 1:2 2,525 2,525 100 (pg/mL) 1:4 1,263 1,340 106 1:8 631 686 109 β-2M 1:100 0.63 0.63 100 (μg/mL) 1:200 0.31 0.34 106 1:400 0.16 0.17 107 Cystatin C 1:100 249 249 100 (ng/mL) 1:200 125 122 102 1:400 62 56 110 NGAL 1:100 1,435 1,435 100 (ng/mL) 1:200 718 775 108 1:400 359 369 103 Osteopontin 1:100 6,415 6,415 100 (ng/mL) 1:200 3,208 3,275 102 1:400 1,604 1,525 95 TIMP-1 1:100 35 35 100 (ng/mL) 1:200 18 18 100 1:400 8.8 8.8 100 A-1M 1:2000 37 37 100 (μg/mL) 1:4000 18 18 99 1:8000 9.1 9.2 99 THP 1:2000 28 28 100 (μg/mL) 1:4000 14 14 96 1:8000 6.7 7.1 94 TFF-3 1:2000 8.8 8.8 100 (μg/mL) 1:4000 3.8 4.4 86 1:8000 1.9 2.2 86

The results of this experiment demonstrated reasonably linear responses of the sandwich-capture assay to variations in the concentrations of the analytes in the tested samples.

Example 4 Spike Recovery of Analytes Associated with Renal Disorders

To assess the recovery of analytes spiked into urine, serum, and plasma samples by an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were spiked into known urine, serum, and plasma samples. Prior to analysis, all urine samples were diluted 1:2000 (sample: diluent), all plasma samples were diluted 1:5 (sample: diluent), and all serum samples were diluted 1:2000 (sample: diluent).

The concentrations of the analytes in the samples were measured using the methods described in Example 1. The average % recovery was calculated as the proportion of the measurement of analyte spiked into the urine, serum, or plasma sample (observed) to the measurement of analyte spiked into the standard solution (expected). The results of the spike recovery analysis are summarized in Table 5.

TABLE 5 Spike Recovery of Analyte Assay in Urine, Serum, and Plasma Samples Recovery in Recovery in Recovery in Spike Urine Serum Plasma Analyte Concentration Sample (%) Sample (%) Sample (%) Calbindin 66 76 82 83 (ng/mL) 35 91 77 71 18 80 82 73 average 82 80 76 Clusterin 80 72 73 75 (ng/mL) 37 70 66 72 20 90 73 70 average 77 70 72 CTGF 8.4 91 80 79 (ng/mL) 4.6 114 69 78 2.4 76 80 69 average 94 77 75 GST-alpha 27 75 84 80 (ng/mL) 15 90 75 81 7.1 82 84 72 average 83 81 78 KIM-1 0.63 87 80 83 (ng/mL) .029 119 74 80 0.14 117 80 78 average 107 78 80 VEGF 584 88 84 82 (pg/mL) 287 101 77 86 123 107 84 77 average 99 82 82 β-2M 0.97 117 98 98 (μg/mL) 0.50 124 119 119 0.24 104 107 107 average 115 108 105 Cystatin C 183 138 80 103 (ng/mL) 90 136 97 103 40 120 97 118 average 131 91 108 NGAL 426 120 105 111 (ng/mL) 213 124 114 112 103 90 99 113 average 111 106 112 Osteopontin 1,245 204 124 68 (ng/mL) 636 153 112 69 302 66 103 67 average 108 113 68 TIMP-1 25 98 97 113 (ng/mL) 12 114 89 103 5.7 94 99 113 average 102 95 110 A-1M 0.0028 100 101 79 (μg/mL) 0.0012 125 80 81 0.00060 118 101 82 average 114 94 81 THP 0.0096 126 108 90 (μg/mL) 0.0047 131 93 91 0.0026 112 114 83 average 123 105 88 TFF-3 0.0038 105 114 97 (μg/mL) 0.0019 109 104 95 0.0010 102 118 93 average 105 112 95

The results of this experiment demonstrated that the sandwich-type assay is reasonably sensitive to the presence of all analytes measured, whether the analytes were measured in standard samples, urine samples, plasma samples, or serum samples.

Example 5 Matrix Interferences of Analytes Associated with Renal Disorders

To assess the matrix interference of hemoglobin, bilirubin, and triglycerides spiked into standard samples, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were spiked into known urine, serum, and plasma samples. Matrix interference was assessed by spiking hemoglobin, bilirubin, and triglyceride into standard analyte samples and measuring analyte concentrations using the methods described in Example 1. A % recovery was determined by calculating the ratio of the analyte concentration measured from the spiked sample (observed) divided by the analyte concentration measured form the standard sample (expected). The results of the matrix interference analysis are summarized in Table 6.

TABLE 6 Matrix Interference of Hemoglobin, Bilirubin, and Triglyceride on the Measurement of Analytes Matrix Compound Maximum Overall Spiked into Spike Recovery Analyte Sample Concentration (%) Calbindin Hemoglobin 500 110 (mg/mL) Bilirubin 20 98 Triglyceride 500 117 Clusterin Hemoglobin 500 125 (mg/mL) Bilirubin 20 110 Triglyceride 500 85 CTGF Hemoglobin 500 91 (mg/mL) Bilirubin 20 88 Triglyceride 500 84 GST-alpha Hemoglobin 500 100 (mg/mL) Bilirubin 20 96 Triglyceride 500 96 KIM-1 Hemoglobin 500 108 (mg/mL) Bilirubin 20 117 Triglyceride 500 84 VEGF Hemoglobin 500 112 (mg/mL) Bilirubin 20 85 Triglyceride 500 114 β-2M Hemoglobin 500 84 (μg/mL) Bilirubin 20 75 Triglyceride 500 104 Cystatin C Hemoglobin 500 91 (ng/mL) Bilirubin 20 102 Triglyceride 500 124 NGAL Hemoglobin 500 99 (ng/mL) Bilirubin 20 92 Triglyceride 500 106 Osteopontin Hemoglobin 500 83 (ng/mL) Bilirubin 20 86 Triglyceride 500 106 TIMP-1 Hemoglobin 500 87 (ng/mL) Bilirubin 20 86 Triglyceride 500 93 A-1M Hemoglobin 500 103 (μg/mL) Bilirubin 20 110 Triglyceride 500 112 THP Hemoglobin 500 108 (μg/mL) Bilirubin 20 101 Triglyceride 500 121 TFF-3 Hemoglobin 500 101 (μg/mL) Bilirubin 20 101 Triglyceride 500 110

The results of this experiment demonstrated that hemoglobin, bilirubin, and triglycerides, three common compounds found in urine, plasma, and serum samples, did not significantly degrade the ability of the sandwich-capture assay to detect any of the analytes tested.

Example 6 Sample Stability of Analytes Associated with Renal Disorders

To assess the ability of analytes spiked into urine, serum, and plasma samples to tolerate freeze-thaw cycles, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. Each analyte was spiked into known urine, serum, and plasma samples at a known analyte concentration. The concentrations of the analytes in the samples were measured using the methods described in Example 1 after the initial addition of the analyte, and after one, two and three cycles of freezing and thawing. In addition, analyte concentrations in urine, serum and plasma samples were measured immediately after the addition of the analyte to the samples as well as after storage at room temperature for two hours and four hours, and after storage at 4° C. for 2 hours, four hours, and 24 hours.

The results of the freeze-thaw stability analysis are summarized in Table 7. The % recovery of each analyte was calculated as a percentage of the analyte measured in the sample prior to any freeze-thaw cycles.

TABLE 7 Freeze-Thaw Stability of the Analytes in Urine, Serum, and Plasma Period Urine Sample Serum Sample Plasma Sample and Recovery Recovery Recovery Analyte Temp Concentration (%) Concentration (%) Concentration (%) Calbindin Control 212 100 31 100 43 100 (ng/mL) 1X 221 104 30 96 41 94 2X 203 96 30 99 39 92 3X 234 110 30 97 40 93 Clusterin 0 315 100 232 100 187 100 (ng/mL) 1X 329 104 227 98 177 95 2X 341 108 240 103 175 94 3X 379 120 248 107 183 98 CTGF 0 6.7 100 1.5 100 1.2 100 (ng/mL) 1X 7.5 112 1.3 82 1.2 94 2X 6.8 101 1.4 90 1.2 100 3X 7.7 115 1.2 73 1.3 107 GST- 0 12 100 23 100 11 100 alpha 1X 13 104 24 105 11 101 (ng/mL) 2X 14 116 21 92 11 97 3X 14 111 23 100 12 108 KIM-1 0 1.7 100 0.24 100 0.24 100 (ng/mL) 1X 1.7 99 0.24 102 0.22 91 2X 1.7 99 0.22 94 0.19 78 3X 1.8 107 0.23 97 0.22 93 VEGF 0 1,530 100 1,245 100 674 100 (pg/mL) 1X 1,575 103 1,205 97 652 97 2X 1,570 103 1,140 92 612 91 3X 1,700 111 1,185 95 670 99 β-2M 0 0.0070 100 1.2 100 15 100 (μg/mL) 1X 0.0073 104 1.1 93 14 109 2X 0.0076 108 1.2 103 15 104 3X 0.0076 108 1.1 97 13 116 Cystatin C 0 1,240 100 1,330 100 519 100 (ng/mL) 1X 1,280 103 1,470 111 584 113 2X 1,410 114 1,370 103 730 141 3X 1,420 115 1,380 104 589 113 NGAL 0 45 100 245 100 84 100 (ng/mL) 1X 46 102 179 114 94 112 2X 47 104 276 113 91 108 3X 47 104 278 113 91 109 Osteopontin 0 38 100 1.7 100 5.0 100 (ng/mL) 1X 42 110 1.8 102 5.5 110 2X 42 108 1.5 87 5.5 109 3X 42 110 1.3 77 5.4 107 TIMP-1 0 266 100 220 100 70 100 (ng/mL) 1X 265 100 220 10 75 108 2X 255 96 215 98 77 110 3X 295 111 228 104 76 109 A-1M 0 14 100 26 100 4.5 100 (μg/mL) 1X 13 92 25 96 4.2 94 2X 15 107 25 96 4.3 97 3X 16 116 23 88 4.0 90 THP 0 4.6 100 31 100 9.2 100 (μg/mL) 1X 4.4 96 31 98 8.8 95 2X 5.0 110 31 100 9.2 100 3X 5.2 114 27 85 9.1 99 TFF-3 0 4.6 100 24 100 22 100 (μg/mL) 1X 4.4 96 23 98 22 103 2X 5.0 110 24 103 22 101 3X 5.2 114 19 82 22 102

The results of the short-term stability assessment are summarized in Table 8. The % recovery of each analyte was calculated as a percentage of the analyte measured in the sample prior to any short-term storage.

TABLE 8 Short-Term Stability of Analytes in Urine, Serum, and Plasma Storage Urine Sample Serum Sample Plasma Sample Time/ Sample Recovery Sample Recovery Sample Recovery Analyte Temp Conc. (%) Conc. (%) Conc. (%) Calbindin Control 226 100 33 100 7 100 (ng/mL) 2 hr/ 242 107 30 90 6.3 90 room temp 2 hr. @ 228 101 29 89 6.5 93 4° C. 4 hr @ 240 106 28 84 5.6 79 room temp 4 hr. @ 202 89 29 86 5.5 79 4° C. 24 hr. @ 199 88 26 78 7.1 101 4° C. Clusterin Control 185 100 224 100 171 100 (ng/mL) 2 hr @ 173 94 237 106 180 105 room temp 2 hr. @ 146 79 225 100 171 100 4° C. 4 hr @ 166 89 214 96 160 94 room temp 4 hr. @ 157 85 198 88 143 84 4° C. 24 hr. @ 185 100 207 92 162 94 4° C. CTGF Control 1.9 100 8.8 100 1.2 100 (ng/mL) 2 hr @ 1.9 99 6.7 76 1 83 room temp 2 hr. @ 1.8 96 8.1 92 1.1 89 4° C. 4 hr @ 2.1 113 5.6 64 1 84 room temp 4 hr. @ 1.7 91 6.4 74 0.9 78 4° C. 24 hr. @ 2.2 116 5.9 68 1.1 89 4° C. GST- Control 14 100 21 100 11 100 alpha 2 hr @ 11 75 23 107 11 103 (ng/mL) room temp 2 hr. @ 13 93 22 104 9.4 90 4° C. 4 hr @ 11 79 21 100 11 109 room temp 4 hr. @ 12 89 21 98 11 100 4° C. 24 hr. @ 13 90 22 103 14 129 4° C. KIM-1 Control 1.5 100 0.23 100 0.24 100 (ng/mL) 2 hr @ 1.2 78 0.2 86 0.22 90 room temp 2 hr. @ 1.6 106 0.23 98 0.21 85 4° C. 4 hr @ 1.3 84 0.19 82 0.2 81 room temp 4 hr. @ 1.4 90 0.22 93 0.19 80 4° C. 24 hr. @ 1.1 76 0.18 76 0.23 94 4° C. VEGF Control 851 100 1215 100 670 100 (pg/mL) 2 hr @ 793 93 1055 87 622 93 room temp 2 hr. @ 700 82 1065 88 629 94 4° C. 4 hr @ 704 83 1007 83 566 84 room temp 4 hr. @ 618 73 1135 93 544 81 4° C. 24 hr. @ 653 77 1130 93 589 88 4° C. β-2M Control 0.064 100 2.6 100 1.2 100 (μg/mL) 2 hr @ 0.062 97 2.4 92 1.1 93 room temp 2 hr. @ 0.058 91 2.2 85 1.2 94 4° C. 4 hr @ 0.064 101 2.2 83 1.2 94 room temp 4 hr. @ 0.057 90 2.2 85 1.2 98 4° C. 24 hr. @ 0.06 94 2.5 97 1.3 103 4° C. Cystatin C Control 52 100 819 100 476 100 (ng/mL) 2 hr @ 50 96 837 102 466 98 room temp 2 hr. @ 44 84 884 108 547 115 4° C. 4 hr @ 49 93 829 101 498 105 room temp 4 hr. @ 46 88 883 108 513 108 4° C. 24 hr. @ 51 97 767 94 471 99 4° C. NGAL Control 857 100 302 100 93 100 (ng/mL) 2 hr @ 888 104 287 95 96 104 room temp 2 hr. @ 923 108 275 91 92 100 4° C. 4 hr @ 861 101 269 89 88 95 room temp 4 hr. @ 842 98 283 94 94 101 4° C. 24 hr. @ 960 112 245 81 88 95 4° C. Osteopontin Control 2243 100 6.4 100 5.2 100 (ng/mL) 2 hr @ 2240 100 6.8 107 5.9 114 room temp 2 hr. @ 2140 95 6.4 101 6.2 120 4° C. 4 hr @ 2227 99 6.9 108 5.8 111 room temp 4 hr. @ 2120 95 7.7 120 5.2 101 4° C. 24 hr. @ 2253 100 6.5 101 6 116 4° C. TIMP-1 Control 17 100 349 100 72 100 (ng/mL) 2 hr @ 17 98 311 89 70 98 room temp 2 hr. @ 16 94 311 89 68 95 4° C. 4 hr @ 17 97 306 88 68 95 room temp 4 hr. @ 16 93 329 94 74 103 4° C. 24 hr. @ 18 105 349 100 72 100 4° C. A-1M Control 3.6 100 2.2 100 1 100 (μg/mL) 2 hr @ 3.5 95 2 92 1 105 room temp 2 hr. @ 3.4 92 2.1 97 0.99 99 4° C. 4 hr @ 3.2 88 2.2 101 0.99 96 room temp 4 hr. @ 3 82 2.2 99 0.97 98 4° C. 24 hr. @ 3 83 2.2 100 1 101 4° C. THP Control 1.2 100 34 100 2.1 100 (μg/mL) 2 hr @ 1.2 99 34 99 2 99 room temp 2 hr. @ 1.1 90 34 100 2 98 4° C. 4 hr @ 1.1 88 27 80 2 99 room temp 4 hr. @ 0.95 79 33 97 2 95 4° C. 24 hr. @ 0.91 76 33 98 2.4 116 4° C. TFF-3 Control 1230 100 188 100 2240 100 (μg/mL) 2 hr @ 1215 99 179 95 2200 98 room temp 2 hr. @ 1200 98 195 104 2263 101 4° C. 4 hr @ 1160 94 224 119 2097 94 room temp 4 hr. @ 1020 83 199 106 2317 103 4° C. 24 hr. @ 1030 84 229 122 1940 87 4° C.

The results of this experiment demonstrated that the analytes associated with renal disorders tested were suitably stable over several freeze/thaw cycles, and up to 24 hrs. of storage at a temperature of 4° C.

Example 8 Diagnosis of Renal Damage Using Detection of Analytes in Human Urine Samples

To assess the effectiveness of a human kidney toxicity panel to detect renal damage due to disease states, the following experiment was conducted. Urine samples were obtained from healthy control patients (n=5), renal cancer patients (n=4) and “other” cancer patients (n=8) afflicted with lung cancer, pancreatic cancer, liver cancer, or colon cancer. All urine samples were diluted as described in Example 4 and subjected to a sandwich-capture assay as described in Example 1. Urine concentrations of analytes included in a human kidney toxicity panel were measured by the assay, including alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.

FIG. 1 summarizes the urine concentrations of those analytes that differed significantly from control urine concentrations. The urine concentrations of A1M, NGAL, and THP were slightly elevated for the renal cancer patient group and more significantly elevated for the “other” cancer patient group. Urine B2M concentrations appeared to be elevated for both the renal cancer and “other” cancer patient groups, although the BRM concentrations exhibited more variability than the other analyte concentrations shown in FIG. 1.

The results of this experiment demonstrated that panels of analytes detected in urine samples were capable of identifying patients having renal damage resulting from renal cancer and other cancers.

Example 9 Analysis of Kidney Biomarkers in Plasma and Urine from Patients with Renal Injury

A screen for potential protein biomarkers in relation to kidney toxicity/damage was performed using a panel of biomarkers, in a set of urine and plasma samples from patients with documented renal damage. The investigated patient groups included diabetic nephropathy (DN), obstructive uropathy (OU), analgesic abuse (AA) and glomerulonephritis (GN) along with age, gender and BMI matched control groups. Multiplexed immunoassays were applied in order to quantify the following protein analytes: Alpha-1 Microglobulin (α1M), KIM-1, Microalbumin, Beta-2-Microglobulin (β32M), Calbindin, Clusterin, CystatinC, TreFoilFactor-3 (TFF-3), CTGF, GST-alpha, VEGF, Calbindin, Osteopontin, Tamm-HorsfallProtein (THP), TIMP-1 and NGAL.

Li-Heparin plasma and mid-stream spot urine samples were collected from four different patient groups. Samples were also collected from age, gender and BMI matched control subjects. 20 subjects were included in each group resulting in a total number of 160 urine and plasma samples. All samples were stored at −80° C. before use. Glomerular filtration rate for all samples was estimated using two different estimations (Modification of Diet in Renal Disease or MDRD, and the Chronic Kidney Disease Epidemiology Collaboration or CKD-EPI) to outline the eGFR (estimated glomerular filtration rate) distribution within each patient group (FIG. 2). Protein analytes were quantified in human plasma and urine using multiplexed immunoassays in the Luminex xMAP™ platform. The microsphere-based multiplex immunoassays consist of antigen-specific antibodies and optimized reagents in a capture-sandwich format. Output data was given as g/ml calculated from internal standard curves. Because urine creatinine (uCr) correlates with renal filtration rate, data analysis was performed without correction for uCr. Univariate and multivariate data analysis was performed comparing all case vs. control samples as well as cases vs. control samples for the various disease groups.

The majority of the measured proteins showed a correlation to eGFR. Measured variables were correlated to eGFR using Pearson's correlations coefficient, and samples from healthy controls and all disease groups were included in the analysis. 11 and 7 proteins displayed P-values below 0.05 for plasma and urine (Table 9) respectively.

TABLE 9 Correlation analysis of eGFR and variables for all case samples URINE PLASMA Variable Pearson's r P-Value Variable Pearson's r  P-Value Alpha-1- −0.08 0.3 Alpha-1- −0.33

Microglobulin Microglobulin Beta-2- −0.23 0.003 Beta-2- −0.39

Microglobulin Microglobulin Calbindin −0.16 0.04 Calbindin −0.18 <0.02 Clusterin −0.07 0.4 Clusterin −0.51

CTGF −0.08 0.3 CTGF −0.05 0.5 Creatinine −0.32

Cystatin-C −0.42 <0.0001 Cystatin-C −0.24 0.002 GST-alpha −0.12 0.1 GST-alpha −0.11 0.2 KIM-1 −0.17 0.03 KIM-1 −0.08 0.3 NGAL −0.28 <0.001 Microalbumin_UR −0.17 0.03 Osteopontin −0.33

NGAL −0.15 0.07 THP −0.31

Osteopontin −0.19 0.02 TIMP-1 −0.28 <0.001 THP −0.05 0.6 TFF3 −0.38

TIMP-1 −0.19 0.01 VEGF −0.14 0.08 TFF2 −0.09 0.3 VEGF −0.07 0.4 P values <0.0001 are shown in bold italics P values <0.005 are shown in bold P values <0.05 are shown in italics

For the various disease groups, univariate statistical analysis revealed that in a direct comparison (T-test) between cases and controls, a number of proteins were differentially expressed in both urine and plasma (Table 10 and FIG. 3). In particular, clusterin showed a marked differential pattern in plasma.

TABLE 10 Differentially regulated proteins by univariate statistical analysis Group Matrix Protein p-value AA Urine Calbindin 0.016 AA Urine NGAL 0.04 AA Urine Osteopontin 0.005 AA Urine Creatinine 0.001 AA Plasma Calbindin 0.05 AA Plasma Clusterin 0.003 AA Plasma KIM-1 0.03 AA Plasma THP 0.001 AA Plasma TIMP-1 0.02 DN Urine Creatinine 0.04 DN Plasma Clusterin 0.006 DN Plasma KIM-1 0.01 GN Urine Creatinine 0.004 GN Urine Microalbumin 0.0003 GN Urine NGAL 0.05 GN Urine Osteopontin 0.05 GN Urine TFF3 0.03 GN Plasma Alpha 1 Microglobulin 0.002 GN Plasma Beta 2 Microglobulin 0.03 GN Plasma Clusterin 0.00 GN Plasma Cystatin C 0.01 GN Plasma KIM-1 0.003 GN Plasma NGAL 0.03 GN Plasma THP 0.001 GN Plasma TIMP-1 0.003 GN Plasma TFF3 0.01 GN Plasma VEGF 0.02 OU Urine Clusterin 0.02 OU Urine Microalbumin 0.007 OU Plasma Clusterin 0.00

Application of multivariate analysis yielded statistical models that predicted disease from control samples (plasma results are shown in FIG. 4).

In conclusion, these results form a valuable base for further studies on these biomarkers in urine and plasma both regarding baseline levels in normal populations and regarding the differential expression of the analytes in various disease groups. Using this panel of analytes, error rates from adaboosting and/or random forest were low enough (<10%) to allow a prediction model to differentiate between control and disease patient samples. Several of the analytes showed a greater correlation to eGFR in plasma than in urine.

Example 10 Statistical Analysis of Kidney Biomarkers in Plasma and Urine from Patients with Renal Injury

Urine and plasma samples were taken from 80 normal control group subjects and 20 subjects from each of four disorders: analgesic abuse, diabetic nephropathy, glomerulonephritis, and obstructive uropathy. The samples were analyzed for the quantity and presence of 16 different proteins (alpha-1 microglobulin (α1M), beta-2 microglobulin (β2M), calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF) as described in Example 1 above. The goal was to determine the analytes that distinguish between a normal sample and a diseased sample, a normal sample and an obstructive uropathy (OU) sample, and finally, an glomerulonephritis sample from the other disease samples (diabetic nephropathy (DN), analgesic abuse (AA), and glomerulonephritis (GN)).

From the above protein analysis data, bootstrap analysis was used to estimate the future performance of several classification algorithms. For each bootstrap run, training data and testing data was randomly generated. Then, the following algorithms were applied on the training data to generate models and then apply the models to the testing data to make predictions: automated Matthew's classification algorithm, classification and regression tree (CART), conditional inference tree, bagging, random forest, boosting, logistic regression, SVM, and Lasso. The accuracy rate and ROC areas were recorded for each method on the prediction of the testing data. The above was repeated 100 times. The mean and the standard deviation of the accuracy rates and of the ROC areas were calculated.

The mean error rates and AUROC were calculated from urine and AUROC was calculated from plasma for 100 runs of the above method for each of the following comparisons: disease (AA+GN+OU+DN) vs. normal (FIG. 5, Table 11), AA vs. normal (FIG. 7, Table 13), DN vs. AA (FIG. 9, Table 15, AA vs. GN (FIG. 11, Table 17), and AA vs. OU (FIG. 13, Table 19).

The average relative importance of 16 different analytes (alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF) and 4 different clinical variables (weight, BMI, age, and gender) from 100 runs were analyzed with two different statistical methods—random forest (plasma and urine samples) and boosting (urine samples)—for each of the following comparisons: disease (AA+GN+OU+DN) vs. normal (FIG. 6, Table 12), AA vs. normal (FIG. 8, Table 14), DN vs. AA (FIG. 10, Table 16), AA vs. GN (FIG. 12, Table 18), and AA vs. OU (FIG. 14, Table 20).

TABLE 11 Disease v. Normal Standard Mean deviation method AUROC AUROC random 0.931 0.039 forest bagging 0.919 0.045 svm 0.915 0.032 boosting 0.911 0.06 lasso 0.897 0.044 logistic 0.891 0.041 regression ctree 0.847 0.046 cart 0.842 0.032 matt 0.83 0.023

TABLE 12 Disease v. Normal relative analyte importance Creatinine 11.606 Kidney_Injury_M 8.486 Tamm_Horsfall_P 8.191 Total_Protein 6.928 Osteopontin 6.798 Neutrophil_Gela 6.784 Tissue_Inhibito 6.765 Vascular_Endoth 6.716 Trefoil_Factor_(—) 6.703 Cystatin_C 6.482 Alpha_1_Microgl 6.418 Beta_2_Microglo 6.228 Glutathione_S_T 6.053 clusterin 5.842

TABLE 13 AA v. NL Standard deviation Mean of method AUROC AUROC cart 1 0 bagging 1 0 boosting 1 0 lasso 0.998 0.008 ctree 0.998 0.015 random 0.997 0.012 forest svm 0.977 0.033 logistic 0.933 0.092 regression matt 0.873 0.112

TABLE 14 AA v. NL Relative analyte importance Creatinine 17.800 Tissue_Inhibito 9.953 Total_Protein 8.837 Tamm_Horsfall_P 7.379 Cystatin_C 6.237 Kidney_Injury_M 6.174 Beta_2_Microglo 5.915 Neutrophil_Gela 5.761 Alpha_1_Microgl 5.742 Trefoil_Factor_(—) 5.736 Osteopontin 5.561 Vascular_Endoth 5.338 clusterin 4.892 Glutathione_S_T 4.675

TABLE 15 AA v. DN Standard Mean deviation method AUROC AUROC lasso 0.999 0.008 random 0.989 0.021 forest svm 0.988 0.039 boosting 0.988 0.022 bagging 0.972 0.036 logistic 0.969 0.057 regression cart 0.93 0.055 ctree 0.929 0.063 matt 0.862 0.12

TABLE 16 AA v. DN Relative analyte importance Creatinine 17.57 Total_Protein 10.90 Tissue_Inhibito 8.77 clusterin 6.89 Glutathione_S_T 6.24 Alpha_1_Microgl 6.15 Beta_2_Microglo 6.06 Cystatin_C 5.99 Trefoil_Factor_(—) 5.88 Kidney_Injury_M 5.49 Vascular_Endoth 5.38 Tamm_Horsfall_P 5.33 Osteopontin 4.86 Neutrophil_Gela 4.47

TABLE 17 AA v. GN Standard deviation Mean of method AUROC AUROC svm 0.689 0.11 boosting 0.675 0.102 bagging 0.674 0.106 random 0.66 0.096 forest matt 0.631 0.085 cart 0.626 0.089 logistic 0.614 0.091 regression lasso 0.606 0.102 ctree 0.53 0.061

TABLE 18 AA v. GN Relative analyte importance Creatinine 10.780 Alpha_1_Microgl 8.847 Kidney_Injury_M 8.604 clusterin 8.109 Total_Protein 7.679 Glutathione_S_T 7.493 Neutrophil_Gela 6.721 Vascular_Endoth 6.461 Cystatin_C 6.444 Beta_2_Microglo 6.261 Trefoil_Factor_(—) 6.184 Tamm_Horsfall_P 5.872 Tissue_Inhibito 5.690 Osteopontin 4.855

TABLE 19 AA v. OU Standard deviation Mean of method AUROC AUROC random 0.814 0.11 forest bagging 0.792 0.115 svm 0.788 0.112 lasso 0.786 0.118 boosting 0.757 0.117 matt 0.687 0.111 logistic 0.683 0.116 regression cart 0.665 0.097 ctree 0.659 0.118

TABLE 20 AA v. OU Relative analyte importance Total_Protein 11.502 Tissue_Inhibito 9.736 Cystatin_C 9.161 Alpha_1_Microgl 8.637 Trefoil_Factor_(—) 7.329 Osteopontin 7.326 Beta_2_Microglo 6.978 Neutrophil_Gela 6.577 Glutathione_S_T 6.100 Tamm_Horsfall_P 6.066 Kidney_Injury_M 6.038 Vascular_Endoth 5.946 clusterin 4.751 Creatinine 3.854

It should be appreciated by those of skill in the art that the techniques disclosed in the examples above represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense. 

1. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising six antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
 2. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol.
 3. The assay device of claim 2, wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
 4. The assay device of claim 2, wherein the renal disorder comprises obstructive uropathy, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of creatinine, THP, A1M, clusterin, NGAL, and osteopontin.
 5. The assay device of claim 2, wherein the renal disorder comprises obstructive uropathy, wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of creatinine, THP, alpha-1 microglobulin, clusterin, NGAL, and osteopontin.
 6. The assay device of claim 2, wherein the renal disorder comprises glomerulonephritis, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.
 7. The assay device of claim 2, wherein the renal disorder comprises glomerulonephritis, and wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.
 8. The assay device of claim 2, wherein the renal disorder comprises kidney toxicity, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.
 9. The assay device of claim 2, wherein the renal disorder comprises kidney toxicity, and wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.
 10. The assay device of claim 2, wherein the renal disorder comprises diabetic nephropathy, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.
 11. The assay device of claim 2, wherein the renal disorder comprises diabetic nephropathy, and wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.
 12. The assay device of claim 2, wherein the renal disorder comprises kidney transplant rejection and chronic allograft nephropathy, and wherein the panel comprises three or more antibodies having antigenic determinants for analytes selected from the group consisting of BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol.
 13. The assay device of claim 2, wherein the panel of biomarkers comprises ten or more antibodies having antigenic determinants for analytes selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
 14. The assay device of claim 2, wherein the panel of biomarkers has sixteen antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
 15. The assay device of claim 2, wherein the contact surface comprises a substrate capable of immobilizing analytes captured by the antibodies.
 16. The assay device of claim 15, wherein the substrate comprises a porous material selected from the group consisting of paper, nitrocellulose, cellulose, glass, glass fiber mesh, silica gel, synthetic resins, plastic strips, beads, the inner surface of a well, the surface of a microtitration tray, and combinations thereof.
 17. The assay device of claim 2, further comprising a plurality of indicators, wherein one of the plurality of indicators is attached to one of the three or more antibodies
 18. The assay device of claim 2, wherein the plurality of indicators comprises visual indicators and electrochemical indicators.
 19. The assay device of claim 18, wherein the visual indicators are selected from the group consisting of nanoparticulate gold, polyurethane microspheres loaded with dye compounds, latex microspheres loaded with dye compounds, carbon black, fluorophores, phycoerythrin, radioactive isotopes, nanoparticles, and enzymes such as horseradish peroxidase or alkaline phosphatase that react with a chemical substrate to form a colored product.
 20. The assay device of claim 18, wherein the electrochemical indicators are selected from the group consisting of ascorbate, vitamin E, glutathione, polyphenols, catechols, quercetin, phytoestrogens, penicillin, carbazole, murranes, phenols, carbonyls, benzoates, and trace metal ions such as nickel, copper, cadmium, iron, and mercury.
 21. The assay device of claim 2, wherein the assay method comprises electrophoresis, mass spectrometry, protein microarrays, western blot, immunohistochemical staining, enzyme-linked immunosorbent assay methods, and particle-based capture-sandwich immunoassays.
 22. The assay device of claim 2, wherein the renal disorder comprises glomerulonephritis, interstitial nephritis, tubular damage, vasculitis, glomerulosclerosis, acute renal failure, chronic renal failure, nephrosis, nephropathy, polycystic kidney disease, Bright's disease, renal transplant, chronic unilateral obstructive uropathy, chronic bilateral obstructive uropathy, acute unilateral obstructive uropathy, and acute bilateral obstructive uropathy.
 23. The assay device of claim 2, wherein the renal disorder comprises renal damage caused by exposure to secondary agents and conditions including therapeutic drugs, recreational drugs, contrast agents, toxins, nephrolithiasis, ischemia, liver transplantation, heart transplantation, lung transplantation, and hypovolemia.
 24. The assay device of claim 2, wherein the renal disorder comprises renal damage secondary to a primary disease state including diabetes, hypertension, autoimmune diseases including lupus, Wegener's granulomatosis, and Goodpasture syndrome, primary hyperoxaluria, kidney transplant rejection, sepsis, nephritis secondary to infection of the kidney, rhabdomyolysis, multiple myeloma, and prostate diseases.
 25. The assay device of claim 2, wherein the mammal is selected from the group consisting of humans, apes, monkeys, rats, mice, dogs, cats, pigs, and livestock including cattle and oxen.
 26. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising: a. three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol; b. three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; c. three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and d. three or more indicators, wherein each of the indicators is attached to one of the detection antibodies.
 27. The assay device of claim 26, wherein the three or more capture antibodies have antigenic determinants for the analytes selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
 28. The assay device of claim 26, wherein the panel of biomarkers comprises six or more antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
 29. The assay device of claim 26, wherein the panel of biomarkers comprises ten or more antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
 30. The assay device of claim 26, wherein the panel of biomarkers comprises sixteen or more antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
 31. A kit for diagnosing, monitoring, or determining a renal disorder in a mammal, the kit comprising: a. the assay device of claim 2; and b. a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.
 32. The kit of claim 31, wherein the collection apparatus comprises urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, and aspiration needles.
 33. A kit for diagnosing, monitoring, or determining a renal disorder in a mammal, the kit comprising: a. the assay device of claim 26; and b. a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.
 34. The kit of claim 33, wherein the collection apparatus comprises urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, and aspiration needles.
 35. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers having sixteen antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
 36. A platform for diagnosing, monitoring, or determining a renal disorder in a mammal, the platform comprising at least 6 antibodies selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF. 